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Roche Applied Science

MagNA Pure LC Operators Manual


Version 3.0

True Walk-Away Automation

for Nucleic Acid Purification

Roche Applied Science

MagNA Pure LC Operators Manual


Version 3.0

Table of Contents Chapter A: General Overview


Topic See Page

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8 2. Description of the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

2.1 2.2 2.3 2.3.1 2.3.2 2.3.3 2.3.4 2.4 3.1 3.2 3.2.1 3.2.2 3.2.3 3.2.4 3.2.5 4.1 4.2 4.3 4.4 5.1 5.2 5.3 5.4 5.5 6.1 6.2 6.3 6.4 6.5 6.6 7.1 7.2 7.3

General Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 The MagNA Pure LC System Package . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Main Components of the Instrument. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Front View of the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Back View of the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Side View of the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14 Nozzle Head and Clot Detection System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Layout of the MagNA Pure LC Reagent/Sample Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Accessories included in the MagNA Pure LC System Package . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Additional MagNA Pure LC Accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Additional Cooling Blocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 LC Carousel Centrifuge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Barcode Readers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25 Barcode Printer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Paging Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Position of Disposable Plastics on the Reagent/Sample Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31 Description of Disposable Plastics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Disposable Plastics needed for available Purification Protocols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 Basic Steps of the Working Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Isolation of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Isolation of total RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Isolation of mRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 MagNA Pure LC Nucleic Acid Isolation Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 DNA Isolation Kit I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 RNA Isolation Kit I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 RNA Isolation Kit II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 mRNA Isolation Kit I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Total Nucleic Acid Isolation Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Cross Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Typical Instrument Specifications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Specifications of the HEPA filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Required Specifications for PC, Monitor, and Printer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

3. MagNA Pure LC Accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

4. Disposable Plastics for the MagNA Pure LC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

5. Description of the Isolation Technology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

6. System Performance Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

7. Specification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

8. Instrument Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 9. Warnings and Cautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

9.1 9.2 9.3

Important Safeguards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Precautions to prevent Contamination and Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 Position and Meaning of Warning Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

Chapter A: General Overview


Topic See Page

10. Marks of Conformity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 11. Third Party Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 12. Revision History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 13. License . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 14. Warranty. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 15. Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

Chapter B: How To Operate the MagNA Pure LC System


Topic See Page

1. Overview over the Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 2. System Set-Up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

2.1 2.2 2.2.1 2.2.2 2.3 2.3.1 2.3.2 2.3.3 2.3.4 2.3.5 2.4 3.1 3.2 3.3
4.

Switching on the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .74 Log-in and Start Up Procedure for MagNA Pure LC Software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .75 Log-in as User Administrator. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .75 Log-in as Standard User . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .78 Placing the Accessories on the MagNA Pure LC Reagent/Sample Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .81 Decontamination of the Workstation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .82 Use of the Dispo Lockbar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .83 Positioning the Reagent Tub Rack and the Reaction Tip Racks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .84 Positioning the Liquid Waste Funnel and Tip Waste Disposal Slide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .85 Positioning MagNA Pure LC Cooling Blocks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .86 Placing and Changing the Liquid Drop Catcher Disposable. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .87 Opening the Sample Ordering Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .89 Selecting the appropriate Purification Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .95 Specifying Sample Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .99 The Start Information Screen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Placing the Disposable Plastics on the Reagent/Sample Stage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Placing Elution and Storage Cartridge (Stage position 10/11) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Placing the Processing Cartridges (Stage position 9) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Placing the Tip Stands (Stage position 8) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Placing the Reaction Tips (Stage position 3/4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Placing the Reagent Tubs (Stage position 2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sample Cartridge (Stage position 1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Placing the Waste Bag . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Placing the Liquid Waste Bottle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Confirming the correct Setup of the Reagent/Sample Stage . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 105 106 106 107 108 109 111 112 113 114

3. Sample Ordering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

Preparation for Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

4.1 4.2 4.2.1 4.2.2 4.2.3 4.2.4 4.2.5 4.2.6 4.2.7 4.2.8 4.3

5. Monitoring the Purification Run on the Batch Status Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 6. Evaluation of the Purification Outcome on the Result Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Chapter B: How To Operate the MagNA Pure LC System


Topic See Page

7. Creating and Starting a Post Elution Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

7.1 7.2 7.3 7.3.1 7.3.2 7.3.3 7.4 7.5 7.5.1 7.5.2 7.6 7.7 7.8 8.1 8.2 9.1 9.2 9.3 9.3.1 9.3.2 9.4 9.5 9.6

Opening the Post Elution Steps Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 The Post Elution Steps Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 Programming a new Post Elution Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 Selecting MagNA Pure LC Cooling Blocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 Defining Type, Name, and initial Volume of Cooling Block Positions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Programming Post Elution Pipetting Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132 Saving and Loading a Post-Elution Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 Editing a Post Elution Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 Editing the Protocol-DescriptionTable. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 Editing reaction vessel parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 Starting the Post Elution Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 Viewing the Result on the Post Elution Result Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 Generating a LightCycler SAM-file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 Overview over A-Ring Ordering Screen specific functions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 A-Ring Ordering Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 Liquid Waste Discard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 Decontamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 Exchanging Messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 Leaving a Message . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 Viewing a Message . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 Installation of Cooling Blocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 Installation of Purification Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 Log File Backup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

8. A-Ring Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

9. Service Functions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160

Chapter C: Appendix
Topic See Page

1. List of Error Codes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 2. Maintenance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176

2.1 2.2 2.2.1 2.2.2 2.2.3


3. 4. 5. 6.

Preventive Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 Maintenance by the User . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 General Maintenance Measures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 D-Ring Exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 Leakage Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
182 186 187 193

Trouble Shooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . File Types Used in the MagNA Pure LC Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ordering Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Prologue
Thank you for purchasing MagNA Pure LC, the instrument for true walk-away automation of nucleic acid purification from Roche Applied Science ! The introduction of real-time PCR has increased the need for precise, automated and convenient nucleic acid isolation. The MagNA Pure LC Instrument provides the solution for all real-time PCR formats and other applications where rapid, automated precision is a must. The MagNA Pure LC Instrument is the innovative upstream component within the LightCycler System for fully automated high quality nucleic acid purifications. User-friendly, easy-to-use software controls all functions of the MagNA Pure LC Instrument and all steps of purification protocols. The MagNA Pure LC Instrument works in comIntended use of the instrument

bination with the MagNA Pure LC Reagent Kits, which contain all components required to perform a specified nucleic acid isolation. Use this Operators Manual to learn how to operate and maintain the MagNA Pure LC Instrument. The manual contains all the information needed for day-to-day operation of the instrument, including start-up, instrument and software operation, daily maintenance, and troubleshooting.
Important: Before operating the MagNA Pure LC Instru-

ment, be sure to read and understand the warnings, cautions and safety requirements (contained in the Warnings and Cautions section below).

The MagNA Pure LC Instrument is a robotic workstation for:

automated isolation of nucleic acids (DNA, total RNA, mRNA) from different kinds of crude sample material (whole blood, serum, blood cells, culture cells, tissue, bacteria, fungi) by the use of the specially designed MagNA Pure LC reagent kits, for the purpose of life science research only, and automated filling of different kinds of PCR reaction vessels (LightCycler capillaries, 96-well PCR plates, PCR tube strips) with PCR reaction mixes and template nucleic acid, or other reaction tubes, for the making of dilution series, reaction mixes etc.

The MagNA Pure LC system is intended for general laboratory use only. It was neither developed nor validated by the manufacturer for any kind of in-vitro diagnostic application. Any use of the MagNA Pure LC system as a front-end sample preparation system for in-vitro diagnostic test systems like COBAS Amplicor tests is in the sole responsibility of the user. The validation of the combination MagNA Pure LC nucleic acid isolation and in-vitro diagnostic testing must be done by the user, following the relevant national rules.

How to use this manual

Please refer to the following table to orientate yourself through the contents of the manual:
Part/Chapter Chapter A: General Overview Contents Chapter A of the manual describes the content of the MagNA Pure LC system package how the instrument itself is built-up which accessories and disposable plastics are needed to work with the instrument how nucleic acids are isolated by the MagNA Pure LC Instrument

In addition, it presents typical performance data and the specifications of the complete system. Chapter B: How To Operate the MagNA Pure LC Instrument Chapter B of the manual describes how Chapter C: Appendix you get access to the MagNA Pure LC software you prepare the instrument for a purification run you perform a purification run a Post-Elution protocol is programmed and a Post-Elution run is performed general service functions can be performed with the MagNA Pure LC software

Chapter C of the manual presents additional information on maintenance of the instrument and troubleshooting

MagNA Pure LC Operators Manual Version 3.0

Chapter A

General Overview

1.

Introduction
Even more, the MagNA Pure LC Instrument is able to fill automatically LightCycler capillaries, as well as other types of PCR reaction vessels, with purified nucleic acids and PCR master mixes. This demonstrates the extraordinary flexibility of the MagNA Pure LC system and its associated reagent kits.

Magnetism is the underlying principle of the automated nucleic acid isolation performed by the innovative, highly flexible MagNA Pure LC Instrument from Roche Applied Science. The MagNA Pure LC workstation permits automated nucleic acid purification that matches the LightCycler PCR system's high standards for speed, accuracy, and reliability.

See the following table for an overview over the main features of the MagNA Pure LC Instrument:
Feature Variable Sample Number Description 1 to 32 different samples can be processed. Depending on the purifcation protocol used, 32 samples are processed in 50180 min. Broad Range of Sample Material Total nucleic acid, DNA, RNA, and mRNA can be isolated from a broad range of sample material. Depending on the particular reagent kit used nucleic acids can be prepared from:
whole blood serum/plasma blood cells culture cells human or animal tissue bacteria or fungi

Use variable sample volumes (20 1000 l), cell numbers (1 x 103 1 x 107), or tissue amount (1-10 mg) as starting material (depending on the isolation kit and purification protocol used). Walk-away instrument A completely closed housing, automatic detection of clots and reaction tip loss, and sample tracking make the MagNA Pure LC a true walk-away instrument. Filtration, centrifugation, and all other manual steps are eliminated. Cross-contamination avoidance The instrument does not use vacuum pumps and tubing on the Reagent/Sample Stage. Therefore, the risk of cross-contamination is reduced to an absolute minimum. The inside of the housing can easily be cleaned with commonly used disinfectants and decontaminated with a built-in UV-lamp. Post Elution abilities After purifying nucleic acids, the MagNA Pure LC Instrument can also set up your downstream PCR/RT-PCR reactions for further use in the LightCycler real-time PCR system or any other PCR instrument. According to your instructions, the instrument will automatically add nucleic acids and master mixes into LightCycler capillaries (contained in either LightCycler Centrifuge Adapters or the LightCycler Sample Carousel), 96-well PCR plates, or PCR tubes.

MagNA Pure LC Operators Manual Version 3.0

2.
2.1

Description of the Instrument


General Description

The MagNA Pure LC Instrument is a robotic workstation for automated nucleic acid isolation from crude sample material as well as filling of PCR reaction vessels (LightCycler capillaries, 96-well PCR plates, PCR tubes) and reaction tubes (1.5 ml). It consists of the compact benchtop instrument itself and a PC system. The central processing unit of the instrument is

the robotic arm with its 8-nozzle pipette head. With this pipette head, variable sample numbers from 1 to 32 can be processed in one run. In addition, this pipette head has a specialized sensor unit, which is responsible for detection of clots in sample material and loss of reaction tips.

Here is a brief overview of the workflow of a typical processing run with the MagNA Pure LC workstation:
Sample Ordering

After starting the MagNA Pure LC software, the appropriate purification protocol has to be selected by the user according to the starting sample material and the type of nucleic acid to be isolated. The user enters additionally needed protocol parameters and sample data on the Sample Ordering Screen.
Start Information

Type and amount of isolation reagents and disposable plastics needed for the purification run are automatically calculated by the software according to input parameters. This information is displayed on the Start Information Screen.
Placement of disposable plastics and reagents

These Reaction Tips not only transfer the samples, but also serve as reaction vials for the procedure. Within the tips, nucleic acids are bound to magnetic beads, washed free of impurities, and finally eluted from the magnetic beads. Eluted nucleic acids are then transferred into the wells of the Storage Cartridge. They can be directly dispensed into PCR reaction vials, such as LightCycler capillaries, by using the Post Elution function, or stored for further use. During the run, used Reaction Tips are automatically discarded into an attached, autoclavable waste bag. Liquid waste may also be collected in a disposable container at the end of the run.
Post Elution

Isolation reagents are filled into nuclease-free, disposable Reagent Tubs. Reagent Tubs and other disposable plastics, such as Processing Cartridges and Reaction Tips, are placed on the instruments Reagent/Sample Stage. Samples are loaded into the Sample Cartridge.
Batch run

After completion of a nucleic acid purification run, you can instruct the MagNA Pure LC Instrument to transfer the eluted nucleic acid samples into LightCycler capillaries, wells of a PCR plate, or reaction tubes. Also, the setup of PCR master mixes or dilution series can be performed automatically by the instrument.

After the user has confirmed correct placing of reagents and disposable plastics, the instrument automatically performs all remaining steps of the procedure using specially designed nuclease-free, disposable Reaction Tips.

General Overview

2.2

The MagNA Pure LC System Package

The MagNA Pure LC System Package consists of: the MagNA Pure LC Instrument the PC system with monitor the Accessory Kit the Disposables Starter Set

See the following tables for a detailed description of the System Package:
The MagNA Pure LC System Package contains: System component II *Computer workstation configuration may vary from country to country due to component availibility and technical advances. To confirm exact computer configuration, please contact your local Roche Applied Science representative. Operating System Monitor Printer
Windows 2000

System component I

MagNA Pure LC Instrument MagNA Pure LC Operators Manual Accessory Kit including 2 MagNA Pure LC Cooling Blocks MagNA Pure LC Disposable Starter Set, containing all

components needed to completely fill the Reagent/Sample Stage twice Hardware


PC with a Pentium IV Processor* 256 MB SDRAM (Minimum Configuration) 48x CD-ROM Drive Keyboard and PS/2 Mouse

17" Monitor* Hewlett-Packard Color Inkjet Printer*

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MagNA Pure LC Operators Manual Version 3.0

The Accessory Kit contains all necessary accessories for the MagNA Pure LC Instrument, including: *The two MagNA Pure LC Cooling Blocks with the instrument may also be ordered separately. Three additional cooling blocks are available: A cooling block for the LightCycler Sample Carousel, for Reaction Tubes, and for COBAS Amplicor A-Rings. For details please refer to Chapter A, section 3.2.1 and Chapter C, section 7.

Quantity 2 2 2

Item Reagent Tub Rack Reaction Tip Rack MagNA Pure LC Cooling Blocks*, including MagNA Pure LC Cooling Block, LC Centrifuge Adaptors (Cat. No. 2 190 664, 1 cooling block with 32 LightCycler centrifuge adaptors) MagNA Pure LC Cooling Block, 96-well PCR Plate (Cat. No. 2 189 674, 1 cooling block)

1 1 1 One of each 1 1 2 1 2 10

Tip Waste Disposal Slide Waste Bottle Tray Liquid Waste Funnel Power cable (for U.S. outlets) Power cable (for European outlets) PC Connecting Cable PC Circuit Board Installation CD and Floppy Disc Back-Up CD for Windows NT / MagNA Pure LC Wall Spacers D-Rings for Nozzle Head Item MagNA Pure LC Reagent Tubs (small) (medium) Cat. No. * 3 004 066 (150 tubs)

The MagNA Pure LC Disposables Starter Set contains all components needed for two complete fillings of the Reagent/ Sample Stage, including: For details about plastic disposables and placing of disposables on the Reagent/ Sample Stage see Chapter A, section 4 and Chapter B, section 4.2.

Quantity 2 Tubs 8 Tubs 2 Tubs 4 Tubs 12 of each Tub Lid 4 Tub Lids 18 Tub Lid Seals 6 Trays 6 Trays 6 Cartridges 8 Seals 8 Cartridge 8 Tip Stands 2 Bags 2 Bottles 1 Set

MagNA Pure LC Medium Reagent Tub 20 3 004 058 (150 tubs) MagNA Pure LC Medium Reagent Tub 30 3 045 501(50 tubs) MagNA Pure LC Reagent Tubs (large) MagNA Pure LC Tub Lids (small, medium) MagNA Pure LC Tub Lids (large) MagNA Pure LC Tub Lid Seals MagNA Pure LC Reaction Tips (large) MagNA Pure LC Reaction Tips (small) MagNA Pure LC Sample Cartridges MagNA Pure LC Cartridge Seals MagNA Pure LC Processing Cartridges MagNA Pure LC Tip Stands MagNA Pure LC Waste Bag MagNA Pure LC Waste Bottle 3 004 104 (400 seals) 3 004 171 (960 tips, packed 30 x 32) 3 004 180 (960 tips, packed 30 x 32) 3 004 112 (120 cartridges) 3 118 827 (200 seals) 3 004 147 (160 cartridges) 3 004 155 (200 tip stands) 3 004 201 (200 bags) 3 004 198 (40 bottles) 3 004 074 (120 lids) 3 004 082 (300 lids) 3 004 040 (120 tubs)

Positioning Frames (one of each; see Chapter B, section 4.2.5 for details)

General Overview

11

2.3

Main Components of the Instrument

2.3.1 Front View of the Instrument

4 7 8 9

5 6

Number 1 2

Instrument Part Housing Front Door

Description Builds up the main body part of the instrument. Consists of painted sheet metal and protects from electromagnetic influences, chemicals, and UV-light. Movement of the robotic arm is only possible after the instruments door is closed and locked. This is to avoid handling inside the instrument which could cause injuries. The door is impervious to UV-light from inside the instrument, in case the decontamination function is activated, but permits good view to the inside. Reagents, disposable plastics, and samples are protected from contamination from the outside. Lockage of the door is controled by the software.

Robotic arm

The robotic arm horizontally moves over the Reagent/Sample Stage (x- and y-direction), thereby transporting the Reaction Tips to the required position of pipetting or tip load/discard. It controls the upward and downward movement of the nozzle head (z-direction), the mechanics for the pipetting process, and the movement of the magnetic plate.

UV lamp

The UV lamp (emmision 260 nm) permits decontamination of the Reagent/Sample Stage from nucleic acids. It is controlled by the software. If the front door is opened, it is automatically switched off. See Chapter B, section 9.2 for details about the decontamination function.

Nozzle Head and Magnetic Plate

The nozzle head performs all pipetting steps. Its 8 nozzles can hold up to eight Reaction Tips per time. The nozzle pitch is 9 mm, which is suitable for pipetting into the wells of a standard 96-well PCR-plate. In addition, the nozzle head contains the Tip-loss & Clot-detection System. The magnetic plate contains a permanent magnet and is movable forward and backward. It moves backward when the nozzle head takes up Reaction Tips. After tip uptake it moves forward to a position under the reaction tips. On top of the magnetic plate a channel is located into which a disposable plastic (Reagent Tub Lid Seal) is placed. continued on next page

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MagNA Pure LC Operators Manual Version 3.0

This serves as a Liquid Drop Catcher, which protects from spreading of liquid drops, possibly left on the outside of a reaction tip, during horizontal movement of the robotic arm over the Reagent/Sample Stage. For separation of magnetic beads, which bind the nucleic acids during the isolation process, from the lysate/buffer the magnetic plate moves from behind near to the Reaction Tips, thereby fixing the magnetic beads at the inner surface of the Reaction Tips. Distance between magnet and Reaction Tips is controlled by the software. Note: For cleaning of the magnetic plate, wipe its surface with 70% ethanol using a smooth cloth. 6 7 Status Indicator LEDs Reagent/Sample Stage These four LEDs indicate the current status of the instrument (see Chapter B, section 2.1 for details) The Reagent/Sample Stage provides the positions for uptake of the Sample Cartridge, Reagent Tubs placed in the Reagent Tub Rack, Reaction Tip Racks, Processing Cartridges, and Tip Stands. It also has a Heating Unit for uptake of the Elution Cartridge, and two Cooling Units for uptake of the Storage Cartridge and MagNA Pure LC Cooling Blocks used during Post Elution. See Chapter A, section 2.4 for details. 8 Liquid Waste Bottle Compartment A small door covers the compartment into which the Liquid Waste Discard Bottle is placed. The autoclavable Liquid Waste Discard Bottle optionally can take up liquid waste left in the Processing Cartridges after the completion of a purification run. The liquid level inside the bottle can be seen through the small window. See Chapter B, section 2.3.4 and Chapter B, section 4.2.8 for further details. 9 Tip Waste Disposal Slide The Tip Waste Disposal Slide holds the Tip Waste Bag, which is fixed by a magnetic holder. Through the Tip Waste Disposal Slide used Reaction Tips are discarded from inside the instrument into the Tip Waste Bag. See Chapter B, section 4.2.7 for details.

2.3.2 Back View of the instrument


1

Description Back Side


Number 1 Instrument Part Exhaust fans with HEPA filter Description The upper housing part is cooled by air. The instrument has an air inlet on one instrument side with dust filters. Air is blown out on the back side by two exhaust fans through a HEPA (glassfiber) filter. This HEPA filter holds back aerosols. See Chapter A, section 7.2 for specifications of the HEPA filter. Note: The MagNA Pure LC Instrument is not a fully airtight device. There is an air flow under the platform. Although total air flow from the platform is to go out through the HEPA filter, it cannot be guarenteed that the platform-atmosphere never goes beyond the platform area and passes out without filtration by HEPA. 2 Wall Spacers The wall spacers are inserted during installation of the instrument. They ensure sufficient spacing between the instruments back side and the wall, which is important for proper cooling of the instrument.

General Overview

13

2.3.3 Side View of the instrument

Main Body: Left Side

3 4

1 2 5

Description Left Side Number 1 2 3 4 5 Instrument Part Air inlet Main Switch Sockets Main Switch Socket for power cable Fuses Description Both sides of the instrument have air inlets. Dust filters protect the inside of the instrument from dust particles. Air is blown out by a fan through the bottom base plate. detailed description see below Switch to turn the instrument on or off. Takes up the power cable. Two power cables, one for US wall sockets and one for European wall sockets, are included in the System Package. The MagNA Pure LC Instrument may be used at voltages between 100 V and 240 V. Before using the instrument the correct fuse for the used voltage range has to be inserted (is done by the service engineer only):
Fuse 250 V / 10 A for 115 V AC Fuse 250 V / 5 A for 230 V AC

Connector for PC Communication Cable

The connection cable connects the instrument with the circuit board of the PC.

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MagNA Pure LC Operators Manual Version 3.0

2.3.4 Nozzle Head and Clot Detection System

Nozzle Head

Clot Detection System

Number 1

Instrument part Nozzle

Function Each nozzle takes up one reaction tip. The nozzle head has 8 nozzles with a pitch of 9mm, which is suitable for pipetting standard PCR 96-well plates. The nozzle head also has a sensor which is activated whenever the nozzles or Reaction Tips attached to the nozzles hit an object. The instrument will then stop operation.

D-Ring

Reaction Tips are fixed to the nozzles by the D-rings through elastic force. Simultaneously they seal the inner compartment of the Reaction Tip airtight to avoid leakage and to ensure precise pipetting.

3 4

Ejector Magnetic Plate

By moving the ejector downward Reaction Tips are removed from the nozzles. During tip uptake the ejector ensures even vertical alignment of Reaction Tips. see Chapter A, section 2.3 for details.

Tip-loss/Detection System Hardware 5 6 LED Photodiode lightsensor The Tip-Loss & Clot Detection System is located in the nozzle head of the robotic arm. It uses LEDs with a wavelength of 650 nm (red light) as light source, and a photodiode as light sensor, additionally equipped with a highpass filter to cut UV-light. After Reaction Tip uptake the cylindrical upper part of the tips is located between LED and photosensor, at a position shortly below the aerosol filter within the reaction. Each nozzle has its own pair of LED and sensor. The analog signal of the photosensor is amplified and connected to an analog-digital converter with a certain count range. The amplifier is adjusted in such a way that measurement of no light (dark) results in 0 counts and measurement of direct light from LED to sensor (i.e. if no tip is picked up) results in maximum counts.

General Overview

15

Software For detection of Reaction Tip loss or clotting of samples the system compares the measured light signal before and after performing an action (i.e. tip uptake or discard, liquid aspiration or dispensing). The following situations are possible:
After pickup of an empty Reaction Tip by the nozzle head the tip-loss/clot detection system expects a decrease of the signal by

a certain number of counts. This is due to the light absorbance properties of the Reaction Tip material.
After tip discard the tip-loss/clot detection system expects an increase of the signal by a certain number of counts. After aspiration of a clear buffer into a Reaction Tip, an increase of the signal is expected. This is due to the light focussing and

collecting properties of clear liquids in a cylindrical body, such as a Reaction Tip (the liquid acts like a cylindrical lens)
After aspiration of a sample lysate into a Reaction Tip an additional decrease of the signal compared to an empty tip is expected.

Important: Correct function of the tip-loss/clot detection system is only guaranteed by using the reagents in the MagNA Pure LC nucleic acid isolation kits, otherwise malfunction of the system may occur. Note: Ambient light (especially sunlight) in the laboratory room may cause problems for the tip-loss/clot detection system. Sun light consists of the complete spectrum of all wavelengths with high intensities. If sunlight falls directly onto the Reagent/Sample Stage of the instrument light reflections may reach the photosensors of the tip-loss/clot detection system, leading to a signal overload condition of the amplifiers. Therefore the instrument should be placed in the laboratory in such a way that direct sun light cannot reach the instrument. On the other hand notice that light from incandescent lamps or fluorescent lamps of normal light intensity will not affect the tip-loss/clot-detection system, as long as the light does not shine directly onto the Reagent/Sample Stage. The light intensity in the laboratory should not be higher than 2.5 kLux.

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MagNA Pure LC Operators Manual Version 3.0

2.4

Layout of the MagNA Pure LC Reagent/Sample Stage

The Reagent/Sample Stage is the working area of the instrument, where the purification process takes place. Here the various disposable plastics and reagents as well as the samples are placed. For this purpose it has positions for uptake of disposable racks (Reagent Tub Rack, Reaction Tip Rack) and for uptake of disposable plastics (Sample Cartridge, The Reagent/Sample Stage is subdivided in three main parts:
Name Prologue Unit Function

Processing Cartridge, Tip Stands). It also has a Heating Unit, where the nucleic acid elution process takes place, and two Cooling Units, for uptake of the eluted nucleic acid into the Storage Cartridge, and Cooling Blocks, needed for the Post Elution process.

Provides the instrument with


isolation reagents (in Reagent Tubs/Reagent Tub Rack) Reaction Tips (in Reaction Tip Tray Racks) samples (in the Sample Cartridge)

From here the robotic arm takes up Reaction Tips. During the Prologue phase of the purification run, isolation reagents from the MagNA Pure LC reagent kits are distributed into the wells of the Processing Cartridges. With the start of the actual purification process samples are lysed in the Sample Cartridge (if not provided pre-lysed) and lysed samples are then transferred to the Processing Cartridge. Processing Unit Here Processing Cartridges and Tip Stands are placed. In the Processing Cartridges the actual purification process takes place. Tip Stands serve as parking positions for Reaction Tips during incubation times. Elution & Post Elution Unit Contains the Heating Unit and the small Cooling Unit 1 for uptake of Elution and Storage Cartridge, respectively. In the Elution Cartridge nucleic acids bound to magnetic beads are eluted into Elution Buffer by heating. Eluted nucleic acids are then transferred into the Storage Cartridge, which is cooled to stabilize purified nucleic acids. The large Cooling Unit 2 takes up various available MagNA Pure LC Cooling Blocks. By applying its Post Elution functions the MagNA Pure LC Instrument can be used as a pipetting robot for automatic setup of PCR reactions.

Prologue unit

Processing unit

Elution & Post Elution unit

General Overview

17

The following figures and tables describe the layout and function of the Reagent/Sample Stage in detail.
Note: When describing positions of accessories and dispos-

able plastics in following sections, they are referred to by their respective numbers displayed in the figures below.
Layout of the empty Reagent/Sample Stage

3a

4a

8a

10a

2a 11a

1a

12a

7 6
Layout of the filled Reagent/Sample Stage

5a

9a

3b

4b

8b

10b

2b

11b

1b 12b

9b

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Graphical presentation of the Layout of the Reagent/Sample Stage:

5b

3c

8c

10c

11c 2c

1c

12c

4c
Position Prologue Unit 1 Sample Cartridge Reagent/Sample Stage Part Description

9c

The Sample Cartridge (1b) is placed into the position (1a). The information about the Sample Cartridge is found in the Samples text box (1c) of the Stage Layout Graphic *. Into the wells of the Sample Cartridge the sample material is prepipetted by the user.

Reagent Tubs

The Reagent Tub Rack (2b) is placed into position 2a. It holds the Reagent Tubs, which take up reagents and buffers from the MagNA Pure LC nucleic acid isolation kits. Four different types of Reagent Tubs are available: Large, medium M20, medium M30, and small**. The type of Reagent Tub, that has to be placed in the Reagent Tub Rack, and the type and volume of buffer to be filled in the Reagent Tubs is displayed on the Stage Layout Graphic (2c). See Chapter B, section 4.2.5 for details how to fill and place Reagent Tubs on the Reagent/ Sample Stage.

Large Reaction Tips

The rack for the Large Reaction Tips is placed into position 3a. The rack is filled with Large Reaction Tips pre-packed in tip trays (3b), according to the information displayed on the Stage Layout Graphic (3c). Large Reaction Tips and their rack and trays are colored blue. See Chapter B, section 4.2.4 for details how to place Large Reaction Tips on the Reagent/ Sample Stage.

Small Reaction Tips

The rack for Small Reaction Tips is placed into position 4a. The rack is filled with Small Reaction Tips pre-packed in tip trays (4b), according to the information displayed on the Stage Layout Graphic (4c). Small Reaction Tips and their rack and trays are colored yellow. See Chapter B, section 4.2.4 for details how to place Small Reaction Tips on the Reagent/ Sample Stage. continued on next page

General Overview

19

Dispo(sable) Lockbar

The Dispo Lockbar holds the disposable plastics in their correct positions on the Reagent/ Sample Stage. Sample Cartridge, Reagent Tub Rack, and Reagent Tip Racks can only be placed after opening the Dispo Lockbar. Start of a purification run is only possible after closing the Dispo Lockbar. The status of the Dispo Lockbar is indicated by the Lock Bar Status Indicator on the Stage Layout Graphic (5b). Used Reaction Tips are discarded through the Tip Waste Disposal Hole and the Tip Waste Disposal Slide into the Tip Waste Disposal Bag. Buffers and reagents left in the wells of the Processing Cartridges after completion of a purification run can optionally be discarded into the Liquid Waste Bottle. The Liquid Waste Funnel leads the liquid waste into the waste bottle.

6 7

Tip Waste Disposal Hole Liquid Waste Funnel

Processing Unit 8 Tip Stands There are 5 positions (8a) on the Reagent/Sample Stage for Tip Stands (8b), which are used to store Reaction Tips temporarily, in case they are not in use. Behind each of the four Processing Cartridges there is one accompanying Tip Stand. During a purification run these Tip Stands temporarily park Reactions Tips, which are currently not in use due to an incubation period. An additional fifth Tip Stand is located behind the Tip Stand of Processing Cartridge #4: This stores reagent dispensing tips, if sample numbers not divisable by eight are processed. Number and position of Tip Stands is displayed on the Stage Layout Graphic (8c). See Chapter B, section 4.2.3 for details on how to place Tip Stands on the Reagent/Sample Stage. 9 Processing Cartridge The Processing Cartridges (9b) are placed into the four identical positions in the center of the Reagent/Sample Stage (9a). The actual purification process takes place in the wells of the Processing Cartridges. The number of Processing Cartridges depends on the sample number: For every 8 samples one Processing Cartridge is needed. Amount and position of Processing Cartridges is shown on the Stage Layout Graphic (9c). See Chapter B, section 4.2.2 for details on how to place Processing Cartridges on the Reagent/Sample Stage.

Elution & Post Elution Unit 10 Heating Unit The Heating Unit (10a) is heated to 60C (during stand-by) or to 80-90C (during operation, depending on the purification protocol used) by a Peltier element. An Elution Cartridge (10b) is placed into the Heating Unit. Inside the wells of the cartridge elution of isolated nucleic acids from the magnetic beads in Elution Buffer takes place. Temperature status of the Heating Unit is shown by the Heat Unit Status Indicator on the Stage Layout Graphic (10c). See Chapter B, section 4.2.1 for details on how to place the Elution Cartridge into the Heating Unit. 11 Cooling Unit 1 Cooling Unit 1 (11a) is cooled to 7.5C by a Peltier element. Into Cooling Unit 1 a Storage Cartridge (11b) is placed, which takes up the eluted nucleic acid samples. Temperature status of Cooling Unit 1 is shown by the Cool Unit 1 Status Indicator on the Stage Layout Graphic (11c). See Chapter B, section 4.2.1 for details on how to place the Storage Cartridge into Cooling Unit 1. 12 Cooling Unit 2 Cooling Unit 2 (12a) is cooled to 7.5C by a Peltier element. Cooling Unit 2 is suitable for placing of user exchangable MagNA Pure LC Cooling Blocks (12b: e.g., LC Carousel Cooling Block), which are used during the Post Elution process for setup of PCR reactions. Temperature status of Cooling Unit 2 is shown by the Cool Unit 2 Status Indicator on the Stage Layout Graphic (12c). Note: The appropriate MagNA Pure LC Cooling Block is shown on the Stage Layout Graphic (12c) only if a Post Elution protocol was defined during Sample Ordering. See Chapter A, section 3 for a description of MagNA Pure LC Cooling Blocks and Chapter B, section 2.3.5 for a description on how to place them into Cooling Unit 2.

* see chapter B, section 4.1 for details on how to access and use the Stage Layout Graphic ** see chapter A, section 4 for a detailed description of disposable plastics

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3.
3.1.

MagNA Pure LC Accessories


Accessories included in the MagNA Pure LC System Package
1. Reagent Tub Rack (2 Racks) The Reagent Tub Rack is placed into the Prologue Unit of the Reagent/Sample Stage (position 2a). Into the Reagent Tub Rack the Reagent Tubs, which are the storage containers for the isolation reagents, are placed. It has two positions for Large Reagent Tubs and six for Medium/Small Reagent Tubs.

2. Reaction Tip Rack L (large) and S (small) (one of each) Into the Reaction Tip Racks the Reaction Tip Trays are placed. The Reaction Tip Racks are placed into the Processing Unit (positions 3a, 4a) located in the middle of the Reagent/Sample Stage between the Reagent Tub Rack position and the Processing Cartridge positions. The rack for the Small Reaction Tips (yellow) is placed into the front position (4a), while the rack for the Large Reaction Tips (blue) is placed into the rear position (3a). 3. MagNA Pure LC Cooling Block for LightCycler Centrifuge Adapters The MagNA Pure LC Cooling Block, LC Centrifuge Adapters, holds 32 LightCycler Centrifuge Adapters. It enables the automated filling of LightCycler Capillaries placed into the adapters, using the Post Elution function of MagNA Pure LC Instrument. In addition, 15 reaction tubes (1.5 ml Sarstedt screw cap tubes) for uptake of PCR reagents or master mixes can be placed into this Cooling Block. This Cooling Block is placed into Cooling Unit 2 and can be combined with either the MagNA Pure LC Cooling Block, Reaction Tubes or 96-well PCR Plate. 4. MagNA Pure LC Cooling Block for 96-well PCR Plate The MagNA Pure LC Cooling Block, 96-well PCR Plate, holds one 96-well PCR plate or several 0.2 ml 8-strip PCR tubes, suitable for the most commonly used PCR block thermocyclers. These PCR reaction vials can be filled automatically using the Post Elution function of MagNA Pure LC Instrument for the setup of Reverse Transcription or PCR reaction mixes. This Cooling Block is placed into Cooling Unit 2 and can be combined with either the MagNA Pure LC Cooling Block, Reaction Tubes or LightCycler Centrifuge Adapters. See table in Chapter B, section 7.3.1 for types and brands of PCR 96-well plates that can be used in combination with this Cooling Block.

General Overview

21

5. Tip Waste Disposal Slide (stainless steel) The Tip Waste Disposal Slide holds the Tip Waste Bag, which is fixed by a magnetic holder. Through the Tip Waste Disposal Slide used reaction tips are discarded from inside the instrument into the Tip Waste Bag. The Tip Waste Disposal Slide is inserted into the slot (next to the Liquid Waste Bottle compartment) provided in the lower left side of the instruments front panel. See Chapter B, section 2.3.4 for details on placing the Tip Waste Disposal Slide. 6. Waste Bottle Tray The Waste Bottle Tray holds the Liquid Waste Bottle. See Chapter B, section 4.2.8 for details on placing the Liquid Waste Bottle.

7. Liquid Waste Funnel The Liquid Waste Funnel is inserted into position 12 (see Chapter B, section 4.2.8). Through the funnel liquid waste is lead into the waste bottle. See Chapter B, section 2.3.4 for details on placing the Liquid Waste Funnel. Note: Do not autoclave the Liquid Waste Funnel.

8. Power Cables (for US / European outlets) and PC Connecting Cable One power cable is suitable for US wall plug sockets, the other is used for European wall plug sockets. The connection cable connects the PC via the PC Circuit Board and the MagNA Pure LC Instrument.

9. PC Circuit Board The PC Circuit Board is responsible for communication between PC and instrument. It is installed into the PC of the MagNA Pure LC system by a service engineer.

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MagNA Pure LC Operators Manual Version 3.0

10. Installation CD and Floppy Disk; The installation CD-ROM and Floppy Disk contain the MagNA Pure LC software and instrument specific data needed for installation of the software. Note: The software is pre-installed by the service engineer during system installation 11. Wall Spacers The wall spacers are inserted by the service engineer during installation of the instrument. They ensure sufficient spacing between the instruments back side and the wall, which is important for optimal cooling of the instrument.

3.2.

Additional MagNA Pure LC Accessories

3.2.1. Additional cooling blocks

Three additional MagNA Pure LC Cooling Blocks are available and can be ordered separately: The MagNA Pure LC
Cooling Block, LC Sample Carousel holds one

MagNA Pure LC Reagent /Sample Stage. This cooling block can be combined with either the MagNA Pure LC Cooling block, LC Centrifuge Adapters or 96-well PCR Plate. The MagNA Pure
LC Cooling Block, A-Ring is suited

LightCycler Sample Carousel, enabling the automatic filling of capillaries placed into the carousel by the MagNA Pure LC. After placing the LightCycler Sample Carousel loaded with capillaries into the cooling block, it is covered by a cooling plate (note shown in the figure beside). This cooling plate serves for efficient cooling of the capillaries reagent reservoirs. In addition, 16 reaction tubes (1.5 ml Sarstedt screw cap tubes) can be placed on the MagNA Pure LC Cooling Block, LC Carousel for pipetting of PCR master mixes. To move the PCR reaction mixes from the reservoir to the bottom of the capillaries the complete carousel with the inserted capillaries is spun in the specially designed LC Carousel Centrifuge.
The MagNA Pure LC Cooling Block, Reaction Tubes is suitable for pla-

for holding two COBAS Amplicor A-Rings with 12 reaction vessels each. The cooling block provides twotimes 12 holes each for uptake of the A-Ring reaction vessels, and lids. It also has two positions for uptake of the A-Ring support including the barcode. In addition, four large and four small reagent tubes can be placed for uptake of PCR reagents.
Note: If using the MagNA Pure LC Cooling Block, A-Ring

cing 32 reaction tubes (1.5 ml Sarstedt screw cap tubes) on the

for Post Elution pipetting start the associated purification run from the A-Ring Ordering Screen. This provides a specially adapted workflow which ensures proper sample tracking from the primary sample to COBAS Amplicor PCR reaction setup.

General Overview

23

Important: The MagNA Pure LC system is intended for ge-

neral laboratory use only. It was neither developed nor validated by the manufacturer for any kind of in-vitro diagnostic application. Any use of the MagNA Pure LC system as a front-end sample preparation system for in-vitro diagnostic test systems like COBAS Amplicor tests is in the sole responsibility of the user. The validation of the combination MagNA Pure

LC nucleic acid isolation and in-vitro diagnostic testing must be done by the user, following the relevant national rules. These additional Cooling Blocks, as well as those Cooling Blocks included in the MagNA Pure LC System Package and some of the accessories described above, may be ordered separately.

Use the ordering numbers given in the following table when ordering additional accessories:
MagNA Pure LC Cooling Blocks and Accessories, ordering information
See Chapter B, section 7.3.1 for types and brands of PCR plates, MagNA Pure LC Cooling Block for that have been tested and which 96-well PCR Plate* can be used in combination with MagNA Pure LC Cooling Block for the PCR plate cooling block. *included in the MagNA Pure LC System Package LightCycler Sample Carousel MagNA Pure LC Cooling Block for Reaction Tubes MagNA Pure LC Cooling Block for A-Rings Accessories Reagent Reservoir Rack Reagent Tip Rack (Large Tips) Reagent Tip Rack (Small Tips) Tip Waste Slide Liquid Waste Funnel Waste Bottle Tray Catalog Number 3 253 767 3 253 775 3 253 783 3 253 791 3 253 805 3 253 813 Pack Size 1 rack 1 rack 1 rack 1 slide 1 funnel 1 tray 3 201 287 1 cooling block 2 189 666 1 cooling block 2 189 674 2 189 704 Cooling Block MagNA Pure LC Cooling Block for LightCycler Centrifuge Adapters* Catalog Number 2 190 664 Pack Size 1 cooling block with 32 LightCycler centrifuge adapters 1 cooling block 1 cooling block

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MagNA Pure LC Operators Manual Version 3.0

3.2.2. LC Carousel Centrifuge

The LC Carousel Centrifuge is an easy-to-use centrifuge, which contains a specially designed rotor that can hold one LightCycler Sample Carousel. After pipetting the reaction mix into the polypropylene cups of the LightCycler Capillaries, either manually or automatically with the MagNA Pure LC Instrument, the capillaries are sealed with the stoppers, placed into the LightCycler Sample Carousel and the whole setup is then centrifuged for 30 seconds to spin down the reaction mix to the tip of the capillary.
Ordering Information
Product Name 1 centrifuge plus rotor; 230V 1 centrifuge plus rotor; 115V Cat. No. Cat. No. 2 189 682 Cat. No. 3 030 512

Instrument characteristics

Type Hardware Dimensions Weight Capacity Centrifugal force Power source Power consumption

Lab-top Centrifuge 315 W x 380 D x 285 H mm 21 kg LightCycler Sample Carousel with 1 - 32 LightCycler Capillaries 735 x g AC 90 to 152 V or 198 to 254 V, 10 A, 50/ 60 Hz max. 170 W

3.2.3. Barcode Readers

The MagNA Pure LC Barcode Readers are optional accessories to the MagNA Pure LC Instrument. The use of a barcode reader is recommended if you want to read in data of barcoded samples into the respective entry fields of the Sample Ordering Screen (see Chapter B, section 3.3 for details), which eases samples tracking and makes it even more reliable. Furthermore, by use of a barcode reader barcode data can be read in into any appropriate text entry field of the MagNA Pure LC software (e.g., barcode of the Sample Cartridge into the file name entry field during saving of a Sample Ordering File or Result Screen file; see sections B.3 and B.6 for details).

Note: Use of barcode readers is obligatory when applying

the A-Ring workflow, as this requires to read-in the barcodes of primary samples, Sample Cartridge, and COBAS AMPLICOR A-Rings. See Chapter B, section 8 for details. The Barcode Reader VS800 and QuickScan 1000 have been tested and approved for use with the MagNA Pure LC Instrument. The Barcode Readers can be integrated in the MagNA Pure LC System by using the PS2 interface (connection via the keyboard). For installation, please follow the operating instructions of the respective reader.

Ordering Information

Product Name MagNA Pure LC Barcode Reader VS800 MagNA Pure LC Barcode Reader QuickScan 1000

Cat. No. 3 137 830 3 186 580

General Overview

25

MagNA Pure LC Barcode Reader VS800 VS800 is an omnidirectional, comfortable, and compact barcode reader which is able to read barcodes in every direction. Thus, it is not necessary to hold the tube in a certain direction. By using an advanced visible laser diode scanning system, the VS800 offers a large depth-of-field and wide scan area for high-performance, hands-free scanning. It can read a variety of barcodes including labels that are long, truncated, partially obscured, or otherwise difficult to read. It autodiscriminates all popular barcodes, like UPC or EAN.

Instrument Specifications Mechanical

Dimensions: Weight: Cable length:

15.0 x 8.6 x 7.6 cm 363 g 2.4 or 3.5 m extended 4.7 V to 14 VDC; 3 Watts maximum

Electrical

Operating voltage: Power consumption:

Universal Wedge Version: 5.2 V to 14 VDC Decoding Capability UPC A, E / EAN 8,13 / JAN 8, 13 Add-ons: UPC P2, UPC P5, Code 128 Industrial Codes: Code 128, Code 93, Code 39 (variable length; fixed length label stitching), Interleaved 2 of 5, Codabar, MSI / Plessey Autodiscrimate UPC Edge with Add-ons & 5 industrials Optical Light source: 650nm Visible Laser Diode (VLD) Scan system: Rotating polygon Scan pattern: Ominidrectional mode: 20 lines Scan speed: 1,500 lines per second (lps) Scan angles: 5 Resolution (minimum): 5 mils / 0.13 mm Ambient Light tolerance: 2100 lux maximum

MagNA Pure LC Barcode Reader QuickScan 1000 The QS1000 is a standard decode scanner that can be used either as a hand-held scanner or a fixed scanner. The user can simply wave the reader in a natural position across a barcode, which will be scanned automatically. When placed in its flexible, fixed stand the reader can operate as a handsfree scanner. The QS1000 autodiscriminates all major barcode symbologies, and fully supports the UPC/EAN family of add-ons and code extensions. Advanced signal processing provides exceptional performance even on low contrast bar codes.

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MagNA Pure LC Operators Manual Version 3.0

Instrument Specifications Mechanical

Dimensions: Weight: Body (w/o cable): Cable: Collapsed: 51 cm,

21.84 x 6.78 x 5.28 cm 6.2 ozs./176 g 4.4 ozs./120 g Extended: 2.4 m;

Cable length options Collapsed: 206 cm, Extended: 3.5 m Electrical Operating voltage Undecoded: Decoded: Undecoded: Decoded: Undecoded: Decoded: Decoding Capability 4.2 V to 14 VDC 3.8 V to 20 VDC 100mA @5 VDC 120mA @5 VDC 70mA @5 VDC 110mA @5 VDC

Operating current nominal

Idling current nominal

UPC A,E /EAN 8,13 /JAN 8,13 (P2 /P5,Code 128 EAN
add-ons (interface dependent))

Code 128/EAN 128 Code 39 Interleaved 2 of 5 Codabar Optical

Code 93 Pharmacode 39 Standard 2 of 5,IATA

Light source: Visible Laser Diode (VLD), 680 nm (nominal) Scan system: Electromagnetic dithering mirror Scan Rate: Resolution: 42 scans/sec.(nominal) 5 mils (0.13mm)

Scan angle: 46 Ambient light immunity: Artificial light:1,200 foot candles /12912 Lux Sunlight: 8,000 foot candles /86080 Lux

3.2.4. Barcode Printer

MagNA Pure LC software 3.0 offers the possibility to connect a barcode printer for printing of barcode labels. Barcode labels can be used to label the Sample/Storage Catridge, primary sample tubes, the LightCycler Sample Carousel, or COBAS AMPLICOR A-Rings. The respective functions needed for barcode printing are found in the File menus of the Sample Ordering Screen, A-Ring Ordering Screen, Start Information Screen, and Post Elution Steps Screen.

The desktop barcode printers LP/TLP 2844 manufactured by Zebra Technologies Corp., Vernon Hills, IL, USA, have been tested and approved for use with the MagNA Pure LC Instrument.

General Overview

27

Instrument Specifications

Printing

Technology: Direct Thermal (LP 2844) Thermal Transfer (TLP 2844) Resolution: 203 dpi (8 dots/mm) Print Width: 4.09 (104mm) Print Speed: 4 inches per second (102mm/s)

Electrical and Safety

Autoranging external power supply Input: 100-240VAC, 50-60Hz Output: 20VDC, 2.5A

Operating Environment Physical Specifications

Operating temperature: 40F to 104F (5C to 40C) Humidity: 10% to 90% non-condensing Model: Width: Depth: Height: Weight* LP 7.9 (200mm) 8.38 (213mm) 6.7 (170mm) 3.0 lbs. (1.3 kg) TLP 7.9 (200mm) 9.75 (248mm) 6.8 (173mm) 3.2 lbs. (1.4 kg)

Media Specifications

Types: Labels, Tags, Continuous, Fan-Fold Minimum Width: Length: Thickness: Roll O.D.: Roll I.D.: Sensing: Ribbon: Width: Length: Roll O. D.: Types: 1.3 (33mm) --Wax, resin, wax/resin 4.3 (110mm) 2900 (74m) 1.3 (33mm) 1.00 (25.4mm) 0.38 (9.7mm) 0.003 (0.08mm) -1.00 (25mm) Gap, black mark, notch Maximum 4.25 (108mm) 11.00 (279mm) 0.0075 (0.18mm) 5.00 (127mm) 1.50 (38mm)

For further details on the these barcode printers visit http://www.zebra.com/PA/Printers/product_LP2844.htm. Please contact your local Roche Applied Science representative for details on how to purchase these instruments.

3.2.5 Paging Unit

In combination with MagNA Pure LC software 3.0 a paging unit may be installed optionally to the workstation. In case a text message is generated by the software (e.g., warning or error message), this text is communicated to the users pager by the paging unit. Also, the user is automatically notified of completion of a purification run.

The MessageSender paging system offered by JTECH Communications Inc., Boca Raton, FL, USA, has been tested and approved for use with the MagNA Pure LC workstation. For further details on the MessageSender System visit http://www.jtech.com/products/message_sender.htm. Please contact your local Roche Applied Science representative for details on how to purchase this instrument.

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4.

Disposable Plastics for the MagNA Pure LC Instrument


(RT-PCR, PCR) inhibitors. The production process excludes any biological contamination. All the disposable plastics (except for the waste bags and waste bottles) are sterilized by gamma irradiation or E-beam. The units are subdivided and packaged in separate, nuclease-free bags to avoid contamination after opening the box.
Important: The MagNA Pure LC disposable plastics are

For operation the MagNA Pure LC Instrument needs certain disposable plastics. These are used e.g., either for uptake of samples or isolation reagents (Sample Cartridge, Reagent Tubs), or during the actual purification process (Reaction Tips, Processing Cartridges, Elution/Storage Cartridge). The MagNA Pure LC disposable plastics meet the stringent demands of RT-PCR and PCR. They are guaranteed to be inert to most chemicals used in laboratory settings, nuclease-free, pyrogen-free, ATP-free, and free of amplification

intended for single use only. Never use a disposable plastic several times (even after possibly cleaning it). This would inevitably lead to unexpected and unreliable results.

4.1 Overview See the following table for an overview of the available disposable plastics for the MagNA Pure LC Instrument:
Overview over Disposable Plastics for the MagNA Pure LC Instrument MagNA Pure LC Reagent Tubs (small) MagNA Pure LC Medium Reagent Tubs 20 MagNA Pure LC Medium Reagent Tubs 30 MagNA Pure LC Reagent Tubs (large) MagNA Pure LC Tub Lids (small, medium) Reagent containers for storing up to 3.5 ml of MagNA Pure LC reagents on the MagNA Pure LC Instrument Reagent/Sample Stage. Reagent containers for storing up to 20 ml of MagNA Pure LC reagents on the MagNA Pure LC Instrument Reagent/Sample Stage. Reagent containers for storing up to 30 ml of MagNA Pure LC Elution Buffer on the MagNA Pure LC Instrument Reagent/Sample Stage. Reagent containers for storing up to 100 ml of MagNA Pure LC reagents on the MagNA Pure LC Instrument Reagent/Sample Stage. The Tub Lids are placed on the Small Reagent Tub, the Medium Reagent Tub M20, and the Medium Reagent Tub M30 after adding the reagents. The perforated lid ensures that excess liquid is removed from the outer surface of the Reaction Tips. MagNA Pure LC Tub Lids (large) The Tub Lids are placed on the Large Reagent Tub after adding the reagents. The perforated lid ensures that excess liquid is removed from the outer surface of the Reaction Tips. MagNA Pure LC Tub Lid Seals (Liquid Drop Catcher) MagNA Pure LC Cartridge Seals Seals for all sizes of tub lids allowing the storage of unused reagents in the MagNA Pure LC Reagent Tubs. The seal is also used as disposable for the liquid drop catcher. Adhesive film for sealing the MagNA Pure LC Sample Cartridge for storage purposes. 3 118 827 200 seals 3 004 104 400 seals 3 004 074 120 lids 3 004 082 300 lids 3 004 040 120 tubs 3 045 501 50 tubs 3 004 058 150 tubs 3 004 066 150 tubs Product Application Cat.No. Pack Size

General Overview

29

Overview over Disposable Plastics for the MagNA Pure LC Instrument (continued)

MagNA Pure LC Reaction Tips (small)

Tips for pipetting of reagents and for magnetic bead separation. Integrated filters prevent contamination of the nozzles. Tips (5-100 l volume range) are supplied in convenient trays for direct use in the MagNA Pure LC Instrument.

3 004 180

960 tips (30 x 32)

MagNA Pure LC Reaction Tips (large)

Tips for pipetting of reagents and for magnetic bead separation. Integrated filters prevent contamination of the nozzles. Tips (50-1000 l volume range) are supplied in convenient trays for direct use in the MagNA Pure LC Instrument.

3 004 171

960 tips (30 x 32)

MagNA Pure LC Reaction Tips (small) Refill Package MagNA Pure LC Reaction Tips large) Refill Package MagNA Pure LC Sample Cartridges

Tips (5-100 l volume range) are bulk packed in bags for manual refilling of tip trays.

3 004 236

1000 tips

Tips (50-1000 l volume range) are bulk pakked in bags for manual refilling of tip trays.

3 004 228

1000 tips

Cartridge with 4 x 8 separate wells for aliquoting of primary samples, eluting nucleic acids from magnetic beads, and for storing of purified nucleic acid samples. The sample cartridge format is compatible to commonly used 8-channel multi-pipettes

3 004 112

120 cartridges

MagNA Pure LC Processing Cartridges MagNA Pure LC Tip Stands MagNA Pure LC Waste Bottles

Cartridge with 8 x 8 separate wells for the nucleic acid isolation process. Tip Stands are used for the temporary storage of Reaction Tips, during incubation periods of the isolation process, in order to save tips. The Waste Bottle is suitable for optional collection of up to 330 ml of liquid waste, left over on the Reagent/Sample Stage from a purification run.

3 004 147

160 cartridges

3 004 155

200 tip stands

3 004 198

40 bottles

MagNA Pure LC Waste Bags

The Waste Bags are used for the collection of used Reaction Tips. The bags can be autoclaved.

3 004 201

200 bags

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MagNA Pure LC Operators Manual Version 3.0

4.2

Position of Disposable Plastics on the Reagent/Sample Stage

The figure below gives you an overview over positions for all disposable plastics on the Reagent/Sample Stage. For detailed information on the Reagent/Sample Stage and how

to place disposables on it please refer to Chapter A, section 2.4 and Chapter B, section 4.2.

2 (a)

2 (b)

10/11

Position No. * 1 10 11 2 (a) 2 (b)

Name of Disposable MagNA Pure LC Sample Cartridge used as Elution Cartridge used as Storage Cartridge MagNA Pure LC Reagent Tubs (large) MagNA Pure LC Reagent Tub Lids (large) MagNA Pure LC Medium Reagent Tubs (20) MagNA Pure LC Medium Reagent Tubs (30) MagNA Pure LC Reagent Tubs (small) MagNA Pure LC Reagent Tub Lids (small/medium)

Cat. No. Cat. No. 3 004 112

Cat. No. 3 004 040 Cat. No. 3 004 074 Cat. No. 3 004 058 Cat. No. 3 045 501 Cat. No. 3 004 066 Cat. No. 3 004 082 Cat. No. 3 004 171 Cat. No. 3 004 180 Cat. No. 3 004 201 Cat. No. 3 004 198 Cat. No. 3 004 155 Cat. No. 3 004 147

3 4 6 7 8 9

MagNA Pure LC Reaction Tips (large) MagNA Pure LC Reaction Tips (small) MagNA Pure LC Waste Bag MagNA Pure LC Waste Bottle MagNA Pure LC Tip Stand MagNA Pure LC Processing Cartridge

* please refer to chapter A, section 2.4 for numbering of Reagent/Sample Stage positions

General Overview

31

4.3

Description of Disposable Plastics


Product View

MagNA Pure LC Sample Cartridge

Cat. No. Pack Size Product Description

3 004 112 120 cartridges The Sample Cartridge has 32 separate wells with a volume of 1.5 ml, arranged in 4 rows with 8 wells each. To simplify sample tracking the cartridge rows and wells are labeled alphanumerically (rows 1-4, wells A-H). The cartridge is compatible with commonly used 8-channel multi-pipettes. Each cartridge is one-third the size of a microtiter plate. A special arrangement of spacer fins under the back rim of each sample cartridge ensures the cartridge can only be positioned on the Reagent/Sample Stage in the correct orientation. When the cartridge is placed correctly, the letters on the cartridge are oriented toward the back of the Reagent/Sample Stage and the diagonally cut corner is in the upper left position. The Sample Cartridge provides an extension to its left side, the so called Barcode Labeling Area. This Barcode Labeling Area offers the opportunity to attach a barcode label to the cartridge. With MagNA Pure LC software 3.0 it is possible to install a barcode printer to the system and to print Cartridge Barcodes directly from the software. The Print Cartridge Barcode function is accessible from the Sample Ordering as well as the Start Information Screen, and allows printing of a barcode label that can be attached to the Sample/Storage Cartridge. This barcode label can be used to track and identify the Sample and Storage Cartridge of the current experiment (see Chapter B, section 8 for details).

Application

The cartridge is used for:


Aliquoting 1 to 32 primary samples (Sample Cartridge) Eluting nucleic acids from magnetic particles (Elution Cartridge) Storing purified nucleic acid samples (Storage Cartridge)

Stage Position

There are three positions on the Reagent/Sample Stage where the Sample Cartridge can be placed (see figure on Chapter A, section 2.4):
Sample Cartridge Tray (position 1a, for use as Sample Cartridge) Heating Unit (position 10a, for use as Elution Cartridge) Cooling Unit I (position 11a, for use as Storage Cartridge)

Important

Ensure correct placement of the cartridge. Incorrectly inserted cartridges will strike the Nozzle Head as it passes.

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MagNA Pure LC Cartridge Seal

Cat. No. Pack Size Product Description


Applicaton

3 118 827 200 seals (100 sheets with 2 seals per sheet) The Cartridge Seal is an adhesive film used for sealing the MagNA Pure LC Sample Cartridge for storage purposes. The Cartridge Seal is applied on the cartridge in order to store the nucleic acids in the cartridge. The film protects the isolated nucleic acids against evaporation and contamination. It allows airtight sealing of the Sample Cartridge within a temperature range of -70C to room temperature. The Cartridge Seal has been tested for the absence of nucleases and inhibitors. The film can be removed by careful withdrawal from the cartridge. Individual samples can be taken out of the cartridge by cutting the film above the respective well. The cartridge can then be re-sealed with a new Cartridge Seal.

Important

Do not remove the Cartridge Seal too abruptly from the Sample Cartridge, because this may lead to cross contamination by creating aerosols and carrying-over from well to well.

General Overview

33

MagNA Pure LC Reagent Tub small medium M20 medium M30

Product view

small Cat. No. Pack Size Product Description 3 004 066 150 tubs Reagent capacity of the tubs:
Small, up to 3.5 ml; Medium (M20), up to 20 ml. Medium (M30), up to 30 ml.

medium M20 3 004 058 150 tubs

medium M30 3 045 501 50 tubs

Positioning fins at each side are designed to ensure the tubs fit correctly in the slots of the reagent tub rack. Application The tubs act as reagent reservoirs on the Reagent/Sample Stage. After filling each tub with the appropriate volume of reagent, close it with a Reagent Tub Lid (small/medium). Stage Position There are six positions at the front of the Reagent Tub Rack for either small or medium tubs (see figure on in Chapter B, section 4.2.5). The remaining two positions on the rack are designed for large tubs. The arrangement of the different tubs and the reagent volumes required depends on the particular purification protocol. Note It is recommended to place Reagent Tubs into the Reagent Tub Rack and to fill reagents into the tubs outside the instrument. See Chapter B, section 2.3.3 and Chapter B, section 4.2.5 for details how to place Reagent Tubs and the Reagent Tub Rack. Important Important While the Small and Medium Reagent Tubs show the same cross section, they differ in depth of the buffer reservoir.
If a Reagent Tub with a smaller volume than expected by the selected puri-

fication protocol is placed into the rack, the Reaction Tips will hit the bottom of the tub, and the machine will stall.
Placement of a Reagent Tub with larger volume capacity then expected

could lead to incorrect pipetting because the Reaction Tips may not immerse deep enough.

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MagNA Pure LC Reagent Tub (large)

Product View

Cat. No. Pack Size Product Description

Cat. No. 3 004 040 120 tubs The MagNA Pure LC Reagent Tub (large) is a reagent container for loading up to 100 ml of MagNA Pure LC reagents on the MagNA Pure LC Reagent/Sample Stage. Positioning fins at each side are designed to ensure the tubs fit correctly in the slots of the reagent tub rack. The V-shaped bottom reduces the dead volume to a minimum. Markings (in 10 ml graduations) and numbers (from 20 to 100 ml, in 20 ml graduations) on the tubs help the user estimate the volumes of reagent in the tubs.

Application

The tubs act as reagent reservoirs on the Reagent/Sample Stage. After filling each tub with the appropriate volume of reagent, close it with a Reagent Tub Lid (large).

Stage Position

There are two positions at the rear of the Reagent Tub Rack for large Reagent Tubs (see Chapter B, section 4.2.5). The remaining six places on the rack are designed for small and medium tubs. The arrangement of the different tubs and the reagent volumes required depend on the isolation protocol.

Note

It is recommended to place Reagent Tubs into the Reagent Tub Rack and to fill reagents into the tubs outside the instrument. See Chapter B, section 2.3.3 and Chapter B, section 4.2.5 for details how to place Reagent Tubs and the Reagent Tub Rack.

General Overview

35

MagNA Pure LC Reagent Tub Lid (small/medium)

Product View

Cat. No. Pack Size Product Description Application

Cat. No. 3 004 082 300 lids Each Reagent Tub Lid has eight holes. Flexible slits are arranged around each hole. The MagNA Pure LC Tube Lid (small, medium) is placed on the small and medium MagNA Pure Reagent Tubs after filling with reagents to reduce loss of reagent volume by evaporation. The perforated lid ensures that potential liquid and bubbles from the outer surface of the tip are removed.

Note Important

If you want to store any remaining reagent in a Reagent Tub, seal it with a Tub Lid Seal. Some of the buffers contained in the MagNA Pure LC Reagent Kits contain alcohol. Therefore, it is necessary to close the Reagent Tubs with a Tub Lid to avoid evaporation. Evaporation in turn may lead to change of buffer composition or volume, which might impair the purification result.

MagNA Pure LC Reagent Tubs Lid (large)

Product View

Cat. No. Pack Size Product Description Application

Cat. No. 3 004 074 120 lids Each Reagent Tub Lid has eight holes. Flexible slits are arranged around each hole. The MagNA Pure LC Tub Lid (large) is placed on the large MagNA Pure LC Reagent Tubs after filling with reagents to reduce loss of reagent volume by evaporation. The perforated lid ensures that potential liquid and bubbles from the outer surface of the tip are removed.

Note Important

If you are storing leftover reagent in a Reagent Tub, seal it with a Tub Lid Seal (see next page). Some of the buffers contained in the MagNA Pure LC Reagent Kits contain alcohol. Therefore, it is necessary to close the Reagent Tubs with a Tub Lid to avoid evaporation. Evaporation in turn may lead to change of buffer composition or volume, which might impair the purification result.

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MagNA Pure LC Operators Manual Version 3.0

MagNA Pure LC Tub Lid Seal

Product View

Cat. No. Pack Size Product Description Application

Cat. No. 3 004 104 400 seals The Tub Lid Seal closes both sizes of Tub Lids, preventing evaporation from the tubs and allowing unused reagent to be stored in the tubs. The MagNA Pure LC Tub Lid Seal is suitable for sealing all types of Reagent Tubs to allow storage of reagents being left over. In addition, the Lid Seal is also used as an insert into the Liquid Drop Catcher, which avoids contamination of the Reagent/Sample Stage during movement of the robotic arm.

Stage Position Note Important

Tub Lid Seals may be inserted into any Reagent Tub Lid (large, small/medium). To use the Tub Lid Seal as an insert into the Liquid Drop Catcher, please follow the instructions on in Chapter B, section 2.4. The Tub Lid Seal does not close the Reagent Tubs airtight. As a result, it is recommended to store reagents being left over after a purification run only for hours or overnight, but not for days. Evaporation of liquid may lead to change of buffer composition, thereby causing incorrect purification results. Reagents must be stored at room temperature to avoid cristallization, except Proteinase K solution, which must be stored at 4C. Before starting a purifcation run make sure that all reagents are equilibrated to room temperature. Magnetic particles should never be stored in the Reagent Tubs, because they tend to sediment and clump. Only fill the amount required for one purifica-tion run. When placing a stored reagent/buffer contained in a Reagent Tub back onto the Reagent/Sample Stage for the next purification run, remove the Lid Seal. If this is not done, the Reaction Tips will hit the seal during pipetting which will cause the instrument to stall the purification run.

General Overview

37

MagNA Pure LC Processing Cartridge

Product View

Cat. No. Pack Size Product Description Application

Cat. No. 3 004 147 160 cartridges The cartridge has 64 wells, arranged in eight rows with eight wells each. Before a nucleic acid isolation run, reagents are dispensed into the Processing Cartridge. Each row contains a specific reagent and each well in the row contains the amount needed for one sample. During the run, Reaction Tips aspirate reagents from the cartridge for the different isolation steps.

Stage Position

There are four positions on the Reagent/Sample Stage for the Processing Cartridges (see Chapter A, section 2.4). Each Processing Cartridge is symmetrical and does not have to be placed in a particular orientation. Ensure that the Processing Cartridges are correctly inserted. Incorrectly inserted cartridges will block the Nozzle Head when it performs the surface scan to check for correct positioning of the disposables.

Caution

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MagNA Pure LC Reaction Tip (large)

Product View

Cat. No. Pack Size Product Description

Cat. No. 3 004 171 960 tips (30 x 32) The MagNA Pure LC Instrument uses two different sizes of Reaction Tips (large and small). Reaction Tips are provided pre-packed in tip trays in sets of 32 tips. The Tip Tray for large Reaction Tips is blue and the one for small Reaction Tips is yellow. Reaction Tips contain an integral filter to prevent contamination of the nozzels. The large Reaction Tips have a capacity of 501000 l. The tips are supplied in convenient trays, 32 tips in each Tip Tray, for direct placement onto the MagNA Pure LC Reagent/Sample Stage.

Application
Stage Position

The MagNA Pure LC Reaction Tips (large) are used for dispensing of the reagents, sample transfer, and for magnetic bead separation. There are three positions on the Reagent/Sample Stage for Large Tip Trays (see Chapter A, section 2.4). Place three blue Tip Trays for large Reaction Tips on the back Tip Rack (which is also blue). The positions for Tip Trays containing Large Reaction Tips are designated (back to front) L1, L2, and L3.

Note

Reaction Tips are supplied either in pre-packed Tip Trays (filled with 32 tips) or packed in bulk in plastic bags (MagNA Pure LC Reaction Tip (large) Refill Pakkage: Cat. No. 3 004 228). The intended use of the Reaction Tips in Refill Packages is to refill the Tip Trays if only a small number of tips are used during a purification run. It is not recommended to fill empty Tip Trays completely with tips from Refill Packages, because this poses the danger of contamination.

Important

Do not autoclave Reaction Tips or Tip Trays.

General Overview

39

MagNA Pure LC Reaction Tip (small)

Product View

Cat. No. Pack Size Product Description

Cat. No. 3 004 180 960 tips (30 x 32) The MagNA Pure LC uses two different sizes of Reaction Tips (large and small). Tips are provided pre-packed tip trays in sets of 32 tips. The Tip Tray for large tips is blue and the one for small tips is yellow. Tips contain integral filters to prevent contamination of the nozzels. The small tips have a capacity of 5 100 l. The tips are supplied in convenient trays, 32 tips each tray, for direct placement onto the MagNA Pure LC Reagent/Sample Stage.

Application

The MagNA Pure LC Reaction Tips (small) are used for pipetting in a Post Elution run and for DNA shearing steps in some purification protocols (e.g., DNA High Perfromance Protocol).

Stage Position

There are three positions on the Reagent/Sample Stage for the Small Tip Trays (see Chapter A, section 2.4). Place three yellow Tip Trays for Small Reaction Tips on the front Tip Rack (which is also yellow). The positions for Tip Trays containing Small Reaction Tips are designated (back to front) S1, S2, and S3.

Note

Tips are supplied either in pre-packed Tip Trays (filled with 32 tips) or packed in bulk in plastic bags (MagNA Pure LC Reaction Tip (small) Refill Package: Cat. No. 3 004 236). The intended use of the Reaction Tips in Refill Package is to refill the Tip Trays if only a small number of tips was used during a purification run. It is not recommended to fill empty Tip Trays completely with tips from Refill Packages, because this poses the danger of contamination.

Important

Do not autoclave Reaction Tips or Tip Trays.

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MagNA Pure LC Operators Manual Version 3.0

MagNA Pure LC Tip Stand

Product View

Cat. No. Pack Size Product Description Application


Stage Position

Cat. No. 3 004 155 200 Tip Stands Each Tip Stand contains eight places for small or large Reaction Tips. Tip Stands are used as temporary storage positions for used Reaction Tips between the isolation steps. This temporary storage function saves tips. There are five slots on the Reagent/Sample Stage for Tip Stands, which are used to store Reactions Tips temporarily, in case they are not in use. Four Tip Stands are located behind each of the four Processing Cartridge positions: These store Reaction Tips during incubation periods of the purification protocol. The fifth Tip Stand is located behind the Tip Stand of Processsing Cartridge #4: During the Prologue phase this Tip Stand stores Reaction Tips, if reagents for sample numbers not divisable by eight are distributed.

Note

The slots for the Tip Stands are not ordinary rectangular holes, but are butteflyshaped. Inside each slot are two metal springs (at the left rear end and right front of the slot). These springs keep the Tip Stand parallel to the X-axis. For details on inserting the Tip Stands into the butterfly-shaped slots on the Reagent/Sample Stage, see Chapter B, section 4.2.3.

General Overview

41

MagNA Pure LC Waste Bag

Product View

Cat. No. Pack Size Product Description

Cat. No. 3 004 201 200 bags When the instrument has finished using a set of Reaction Tips, it automatically discards them into the plastic Waste Bag that fits over the Tip Waste Disposal Slide (at front of the MagNA Pure LC Instrument). The MagNA Pure LC Waste Bags are used for the collection of used tips. They can be autoclaved. The bag fits over the Tip Waste Disposal Slide. For details on installing the Waste Bag on the Tip Waste Disposal Slide, see Chapter B, section 4.2.7. The user has to make sure that a Waste Bag is attached to the Tip Waste Disposal Slide and there is enough space left inside the bag for uptake of Reaction Tips from the next purification run. If infectious sample material is purified, Reaction Tips may be contaminated. If the Waste Bag is crammed with used tips, tips may fall out of the bag or get stucked inside the instrument. This may pose danger of contamination of the laboratory environment or malfunction of the system.

Application
Stage Position

Note
Important

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MagNA Pure LC Waste Bottle

Product View

Cat. No. Pack Size Product Description Application

Cat. No. 3 004 198 40 bottles Plastic bottle for uptake of approx. 330 ml liquid waste. MagNA Pure LC Waste Bottle is used for the optional collection of liquid waste by the MagNA Pure LC Instrument. The Waste Bottle is suitable for collecting 330 ml liquid waste and can be autoclaved. The use of the Liquid Waste Bottle is recommended, in order to prevent the user getting into contact with potentially infectious material or the irritating chaotropic salts of the Lysis Buffer.

Stage Position

The Waste Bottle is placed into the Liquid Waste Bottle Compartment (see Chapter A, section 2.3.1) in a way that the Liquid Waste Funnel is inserted into the opening of the bottle. For details on installing the Waste Bottle on the Reagent/Sample Stage, see Chapter B, section 4.2.8. The liquid level inside the Waste Bottle cannot be determined by the MagNA Pure LC Instrument. If the Waste Bottle is too full, contaminated liquid at first spills from the bottle into the Waste Bottle Tray and then into the instrument. This poses the danger of contamination in the laboratory environment. Liquid level inside the bottle should be checked by the user through the small window of the Liquid Waste Bottle compartment.

Note
Important

General Overview

43

4.4.

Disposable Plastics needed for available Purification Protocols

The tables below give an overview over the kind and amount of disposable plastics needed to process a purification run of 32 samples with the various available purification protocols.
Note: If sample numbers not divisable by 8 are run, an

additional tip stand for parking of Reaction Tips is needed.


Cat. No. Pack Size Fast Protocol DNA Isolation Kit I (3 003 990) High Performance Protocol 0 4 1*) 2 4 2 6 1 3 4 4 1 (optional) 80 80 32 32 1 1 External Lysis Protocol DNA Isolation Kit II (3 186 229) Standard Protocol External Proteinase K Protocol

Product Reagent Tubs Small Reagent Tubs Medium M20 Reagent Tub Medium M30 Reagent Tubs Large Reagent Tub Lids Small/Medium1) Reagent Tub Lids Large Reagent Tub Lid Seals Drop Catcher (=Tub Lid Seal) Sample Cartridges Processing Cartridges Tip Stands Cartridge Seals Reaction Tips large2) or Refill Package Reaction Tips small or Refill Package Waste Bottles3) Waste Bags 3 004 066 3 004 058 3 045 501 3 004 040 3 004 082 3 004 074 3 004 104 3 004 104 3 004 112 3 004 147 3 004 155 3 118 827 3 004 171 3 004 228 3 004 180 3 004 236 3 004 198 3 004 201 150 150 50 120 300 120 400 400 12 160 200 200 960 1000 960 1000 40 200 0 4 1*) 2 4 2 6 1 3 4 4 1 (optional) 80 80 0 0 1 1 0 3 1*) 2 3 2 5 1 3 4 4 1 (optional) 80 80 32 32 1 1 1 4 1*) 2 5 2 7 1 3 4 4 1 (optional) 80 80 0 0 1 1 0 4 1*) 2 5 2 7 1 3 4 0 1 (optional) 80 80 0 0 1 1

1) you need one more Reagent Tub Lid if you choose dilution 2) you need one more row if you have selected the Liquid Waste Discard function 3) only of you have selected the Liquid Waste Discard function *only needed when the optional Liquid Waste Discard is activated.

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Cat. No.

Pack Size

DNA RNA Isolation RNA Isolation Kit Kit I Isolation Kit (3 004 007) III II (3 018 997) (3 264 785)

mRNA Isolation Kit I (3 004 015) Blood Cell Protocol Protocol Blood External Lysis

Product Reagent Tubs Small Reagent Tubs Medium M20 Reagent Tub Medium M30 Reagent Tubs Large Reagent Tub Lids Small/Medium1)
3 004 066 3 004 058 3 045 501 3 004 040 3 004 082 3 004 074 3 004 104 3 004 104 3 004 112 3 004 147 3 004 155 3 118 827 3 004 171 3 004 228 3 004 180 3 004 236 3 004 198 3 004 201 150 150 50 120 300 120 400 400 12 160 200 200 960 1000 960 1000 40 200 0 4 1*) 2 5 2 7 1 3 4 4 1 (optional) 80 80 32 32 1 1 0 5 1*) 2 5 2 7 1 3 4 4 1 (optional) 80 80 32 32 1 1 0 5 0 2 5 2 7 1 3 4 4 1 (optional) 80 80 32 32 40 200 1 4 0 2 5 2 7 1 3 4 0 1 (opt) 80 80 32 32 1 1 1 3 0 2 4 2 6 1 3 4 0 1 (opt) 80 80 32 32 1 1 1 3 0 2 4 2 6 1 3 4 0 1 (opt) 80 80

Reagent Tub Lids Large Reagent Tub Lid Seals Drop Catcher (=Tub Lid Seal) Sample Cartridges Processing Cartridges Tip Stands Cartridge Seals Reaction Tips large2) or Refill Package Reaction Tips small or Refill Package Waste Bottles3)

Waste Bags

Cat. No.

Pack Size

mRNA Isolation Kit II (3 172 627)

mRNA HS Kit (3 262 393)

Total Nucleic Acid Isolation Kit (3 038 505) Serum Plasma Blood Variable External Elution Lysis Volume

Total NA Isolation Kit Large Volume (3 264 793)

Product Reagent Tubs Small Reagent Tubs Medium M20 Reagent Tub Medium M30 Reagent Tubs Large Reagent Tub Lids Small/Medium Reagent Tub Lids Large Reagent Tub Lid Seals Drop Catcher (=Tub Lid Seal) Sample Cartridges Processing Cartridges Tip Stands Cartridge Seals Reaction Tips large2) or Refill Package Reaction Tips small or Refill Package Waste Bottles Waste Bags
3) 1)

3 004 066 3 004 058 3 045 501 3 004 040 3 004 082 3 004 074 3 004 104 3 004 104 3 004 112 3 004 147 3 004 155 3 118 827 3 004 171 3 004 228 3 004 180 3 004 236 3 004 198 3 004 201

150 150 50 120 300 120 400 400 12 160 200 200 960 1000 960 1000 40 200

1 3 1*) 1 4 1 5 1 3 4 4 1 (optional) 80 80 32 32 1 1

0 3 1*) 2 4 2 6

0 5 1*) 2 5 2 7 1

0 5 0 2 5 2 7 1 40 40 50

0 4 0 2 4 2 6 1 3 4 4 1 (opt)

0 5 1 2 6 2 8 1 3 4 0

3 4 4

3 4 4 1 (opt)

80 80 32 32 1 1

80 80 0 0 1 1

48 48 32 32 1 1

48 48 32 32 1 1

48 48 32 32 1 1

1) you need one more Reagent Tub Lid if you choose dilution 2) you need one more row if you have selected the Liquid Waste Discard function 3) only of you have selected the Liquid Waste Discard function *only needed when the optional Liquid Waste Discard is activated.

General Overview

45

5.

Description of the Isolation Technology

5.1 Basic Steps of the Working Procedure

For the isolation of DNA, RNA, and mRNA proprietary magnetic particles and specialized reagent kits are used. Nucleic acids bind to the surface of magnetic particles by two different mechanisms: DNA and total RNA bind to Magnetic Glass Particles (MGPs) in the presence of a chaotropic salt at a pH >7.0. MGPs have a glass (silica) surface and a magnetic core. Nucleic acids are adsorbed to the silica-surface of the MGPs in the presence of isopropanol and high concentrations of chaotropic salts, which remove water from hydrated molecules in solution. Polysaccharides and proteins do not adsorb to the beads and are removed by sequential washing steps. Pure nucleic acids are then eluted from the beads by applying low-salt conditions and heat.

mRNA is bound in a different way. For mRNA isolation, streptavidin-coated magnetic particles are used. The streptavidin binds biotin-labeled oligo(dT) probes, which in turn hybridize to the poly(A)-tails of eukaryotic mRNAs. Once bound to the surface of the magnetic beads, the nucleic acids can be separated from the solution with a magnet. The advantage of the MagNA Pure LC isolation technology is that it requires no centrifugation or any other manual steps. For a description of the basic workflow of the instrument during a purification run and of the detailed isolation steps for the various types of nucleic acids, see the following schematics below.

Step 1 During the Prologue Phase reagents are transported from the Reagent Tubs into the wells of the Processing Cartridge.

Step 2 Samples are lysed in the wells of the Sample Cartridge. The lysates are then transferred to the Processing Cartridge.

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Step 3 According to the reaction steps defined by the selected purification protocol the reaction mix is transported to the next rows of wells containing the subsequently used reagents.

Step 4 The complex of magnetic particles and bound nucleic acids is separated from the solution by applying the magnet. Proteins and other contaminating components are washed away by repeated separation and resuspension steps using various wash buffers.

Step 5 The complex of magnetic particles and bound nucleic acid is transported from the Processing Cartridge to the preset Elution Buffer in the Elution Cartridge. Nucleic Acids are released from the magnetic particles by applying heat and low-salt conditions.

Step 6 The eluted and purified nucleic acids are transferred from the Elution Cartridge into the Storage Cartridge.

General Overview

47

5.2

Isolation of DNA

Isolation step Step A Step B Step C Step D Step E Step F

Description The sample material, e.g., whole blood or cells, is placed into the wells of the Sample Cartridge. Addition of Lysis/Binding Buffer results in complete cell lysis and DNA is released. Proteins are denatured. Proteinase K is added to the samples and digestion of cellular proteins starts. DNA binds to the silica surface of the added MGPs due to the chaotropic salt conditions, isopropanol, and the high ionic strength of the Lysis / Binding Buffer. MGPs with bound DNA are magnetically separated from the residual lysed sample. MGPs with bound DNA are washed repeatedly Wash Buffers to remove unbound substances like proteins (nucleases), cell membranes, PCR inhibitors like heparin or hemoglobin, and to reduce the chaotropic salt concentration.

Step G Step H

MGPs with bound DNA are magnetically separated from the Wash Buffer containing residual sample debris. Purified DNA is eluted at 70C in the wells of the Elution Cartridge.

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5.3

Isolation of total RNA

Isolation step Step A Step B Step C Step D Step E Step F

Description The sample material, e.g., whole blood, blood cells or culture cells, is put into the wells of the Sample Cartridge. Addition of Lysis/Binding Buffer results in complete cell lysis and RNA is released. RNases are denatured and inactivated. RNA binds to the silica surface of the added MGPs due to the chaotropic salt conditions, isopropanol, and the high ionic strength of the Lysis / Binding Buffer. Incubation with DNase results in pure RNA without DNA contamination. RNA is rebound to the particles by addition of Isopropanol. MGPs with bound RNA are then magnetically separated from the residual lysed sample. MGPs with bound RNA are washed repeatedly Wash Buffers to remove unbound substances like proteins (nucleases), cell membranes, PCR inhibitors like heparin or hemoglobin, and to reduce the chaotropic salt concentration.

Step G Step H

MGPs with bound RNA are magnetically separated from the Wash Buffer containing residual sample debris. Purified RNA is eluted at 70C in the wells of the Elution Cartridge

General Overview

49

5.4

Isolation of mRNA

Isolation step Step A Step B

Description In the case of starting with whole blood, the sample is pipetted directly into the Sample Cartridge well. Addition of Lysis/Binding Buffer results in complete cell lysis and mRNA is released. If using sample materials like cell suspensions (e.g., WBCs, PBMCs, or culture cells) the preparation of cell pellets and cell lysates has to be done manually. The lysates are then pipetted into the wells of the Sample Cartridge.

Step C

After the lysis step, the sample is diluted with Capture Buffer containing biotin-labeled oligo(dT) probe in Hybrization Buffer. The probe hybridizes to the poly(A) residue of mRNA. After addition of Streptavidin Magnetic Particles (SMPs) the mRNA biotin-labeled oligo(dT) complex is captured and immobilized onto the bead surface during a short incubation period.

Step D Step E Step F

DNA is degraded by incubation in the presence of DNase. SMPs with bound mRNA are magnetically separated from the residual lysed sample. SMPs with bound mRNA are washed repeatedly Wash Buffers to remove unbound substances like proteins (nucleases), cell membranes, PCR inhibitors such as heparin or hemoglobin, and to reduce the chaotropic salt concentration.

Step G Step H

Again SMPs with bound mRNA are magnetically separated from the Wash Buffer containing residual sample debris. Purified mRNA is eluted at 80C in the wells of the Elution Cartridge.

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5.5

MagNA Pure LC Nucleic Acid Isolation Kits

Optimized protocols for nucleic acid isolation (DNA, RNA, mRNA) from various sample materials are pre-installed in the MagNA Pure LC software. All required reagents are supplied (in ready-to-use form) in the MagNA Pure LC Nucleic Acid Isolation Kits.
Nucleic Acid isolated DNA Reagent Kit MagNA Pure LC DNA Isolation Kit I MagNA Pure LC DNA Isolation Kit II (Tissue) MagNA Pure LC DNA Isolation Kit III (Bacteria, Fungi) MagNA Pure LC DNA Isolation Kit - Large Volume 3 310 515

See the following table for MagNA Pure LC Nucleic Acid Isolation Kits currently available:

Catalog Number 3 003 990 192 reactions 3 186 229 192 reactions 3 264 785 192 reactions

Sample Material Whole Blood Blood cells Culture Cells Tissue Bacteria, Fungi

Sample Volume 20 200 l 102-106 cells in 200 l in PBS 1 10 mg 50-100 l

Whole Blood Blood Cells Culture Cells

up to 1000 l whole blood up to 5 x 106 culture cells

96 isolations from 1 ml blood 192 isolations from 300500 l blood 288 isolations from 20200 l blood 192 isolations from blood cells 192 isolations from 5 x 106 culture cells

Total Nucleic Acid (DNA and RNA)

MagNA Pure LC Total Nucleic Acid Isolation Kit MagNA Pure LC Total Nucleic Acid Isolation Kit - Large Volume

3 038 505 192 reactions 3 264 793 192 reactions 3 004 007 192 reactions

Whole Blood Serum Plasma Serum Plasma Whole Blood White Blood Cells Peripheral Blood Mononuclear Cells

50 200 l

200 - 1000 l

total RNA

MagNA Pure LC RNA Isolation Kit I

20 200 L 102-106 cells in 200 l Lysis /Binding Buffer 102-106 cells in 200 l Lysis /Binding Buffer 10 100 l 102-106 cells in 300 l Lysis /Binding Buffer 1 - 10 mg tissue in 300 l Lysis/Binding Buffer 107 cells in 1200 l Lysis/Binding Buffer

MagNA Pure LC RNA Isolation Kit II mRNA MagNA Pure LC mRNA Isolation Kit I (blood, cells) MagNA Pure LC mRNA Isolation Kit II (tissue) MagNA Pure LC mRNA HS Kit

3 018 997 192 reactions 3 004 015 192 reactions 3 172 627 192 reactions 3 267 393 192 reactions

Culture Cells Whole Blood Blood Cells Culture Cells animal or human frozen tissue White Blood Cells Peripheral Blood Mononuclear Cells

General Overview

51

6.

System Performance Data

In this section application data for various MagNA Pure LC reagent kits are shown, which cover important parameters for the characterization of the system performance.
6.1 DNA Isolation Kit I
Pack Size 1 Kit (192 reactions)

Cat. No. 3 003 990

Typical Performance Data


Sample Whole Blood 20 l 50 l 100 l 200 l Cultured Cells K562 103 104 1 x 105 2.5 x 105 5 x 105 1 x 106 Cultured Cells HeLa 103 104 1 x 105 2.5 x 105 5 x 105 1x 106 2.1 5.7 6.0 11.1 1.9 0.1 35.8 30.2 25.8 24.3 24.4 23.8 1.8 3.5 6.0 10.6 1.9 0.1 34.5 30.1 25.8 24.3 23.4 23.1 1,5 3.2 4.6 7.2 1.9 0.1 25.6 24.8 24.7 23.6 Yield [g]
(based on OD260)

Purity
(OD260/280)

CP

Reproducibility Different sample materials were processed with the MagNA Pure LC in replicates of 30. To demonstrate MagNA Pure LCs reproducibility, the eluates were analyzed by LightCycler PCR targeting the Factor V gene (see table). Furthermore, the reproducibility was shown by gel electrophoresis of the eluates and by melting curve analysis as performed for mutation detection for the Factor V gene (20 l blood samples). Similar results were obtained for cultured cells. Reproducibility based on Fac. V LC analysis
Sample Whole Blood 20 l 200 l Culture Cells 105 Hela cells 105 K562 cells 25.6 25.8 1.3 0.8 30 30 25.4 23.0 0.8 2.8 30 30 CP (mean) %CV n

CV: Coefficient of variance reflecting the reproducibility of the combination of MagNA Pure LC and the LightCycler CP: Crossing point

Volume ranges set by the MagNA Pure LC Software


Sample Volume * Elution Volume 20-200 l blood 103-106 cells in 200 l 100 l

Agarose gel analysis of the 30 eluates containing genomic DNA from 20 l blood samples, respectively

Results of the LightCycler PCR analysis of Factor V gene (see table)

Melting curve analysis of the amplified Factor V DNA (constant melting point at 65C)

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Scalability To show the scalability of DNA isolation, blood samples (20 l, 50 l, 100 l, 200 l)) were processed on the MagNA Pure LC Instrument. The amount of DNA was determined by OD 260 measurement. To show the correlation between

quantification by PCR (CPs) and quantification by OD260 measurement the DNA was analyzed by LightCycler PCR targeting the Factor V gene.

PCR with theoretical 100% efficiency results in a difference of crossing points by 3.3, when the amplified amount of DNA increases by factor 10. DNA isolated with the MagNA Pure LC results in a difference of 2.9 crossing points, when analyzed with LightCycler PCR.

MagNA Pure LC isolates high quality genomic DNA

DNA from whole blood, HeLa cells, and K562 cells was isolated with the MagNA Pure LC Instrument (MPLC) and a major competitors manual method (comp). Purified DNA was analyzed by gel electrophoresis. The MagNA Pure LC isolated DNA proved to be superior with respect to integrity and purity (RNA contamination).

General Overview

53

6.2

RNA Isolation Kit I


Pack Size 1 Kit (192 reactions)

Cat. No. 3 004 007

Typical Performance Data


Sample Whole Blood 10 l 20 l 50 l 100 l 27.4 26.7 25.9 25.8 CP

Reproducibility Different sample materials were processed with the MagNA Pure LC Instrument in replicates of 30. To demonstrate MagNA Pure LCs reproducibility, the eluates were analyzed by LightCycler RT-PCR targeting Cyclophilin A transcript (see table).

Reproducibility based on Cyc. A LC analysis


Sample Whole blood ( 100 l) WBCs (from 100 l blood) PBMCs (from 100 l blood) CP 25.4 23.0 26.4 %CV 0.8 2.8 2.3 n 30 30 30

WBCs (isolated from l blood) 50 l 100 l 200 l 400 l 25.7 24.8 24.5 24.1

CV: Coefficient of variance reflecting the reproducibility of the combinati on of MagNA Pure LC DNA isolation and LightCycler RT-PCR.

PBMCs (isolated from l blood) 50 l 100 l 200 l 400 l 27.5 26.7 25.9 25.8

Volume ranges set by the MagNA Pure LC Software


Sample Volume *
(whole blood or 1 x 105 - 5 x 106 PBMCs/WBCs, isolated from blood, in an appropriate buffer)

20-200 l

Elution Volume

100 l
RT-PCR analysis on Cyclophiline A of total RNA, purified from PBMCs, on the LightCycler Instrument (similar results were obtained for whole blood and WBCs).

* Smaller sample volumes need to be filled up with an appropriate buffer to result at least 20 l.

Scalability To show scalability of total RNA isolation the following sample materials

whole blood (10 l, 20 l, 50 l, 100 l) PBMCs isolated from 50 l, 100 l, 200 l, 400 l blood WBCs isolated from 50 l, 100 l, 200 l, 400 l blood

were processed on the MagNA Pure LC Instrument. The eluates were analyzed by LightCycler RT-PCR targeting Cyclophilin A transcript.

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MagNA Pure LC System isolates high quality total RNA RNA was isolated from whole blood (50 l), WBCs, and PBMCs (each isolated from 50 l blood) with the MagNA Pure LC Instrument. Purified RNA was analyzed by gel electrophoresis (SYBR Green II staining). The MagNA Pure LC isolated total RNA proved to be excellent with respect to integrity.

6.3

RNA Isolation Kit II


Pack Size 1 Kit (192 reactions)

Cat. No. 3 018 997

Typical Performance Data


Sample Yield [g]
(based on OD260)

Purity
(OD260/280)

CP

Cultured Cells K562 103 104 1x 105 2.5 x 105 5 x 105 1 x 106 Cultured Cells HeLa 103 104 1 x 105 2.5 x 105 5 x 105 1x 106 1.5 3.6 6.3 10.4 1.9 0.1 26.5 24.6 19.1 18.3 17.5 17.2 2.0 4.2 7.6 12.8 1.9 0.1 31.7 26.3 20.2 19.3 19.1 18.6

Reproducibility Samples of HeLa and K562 culture cells (1 x 105 cells) were processed with the MagNA Pure LC Instrument each in replicates of 30, respectively. To demonstrate MagNA Pure LCs reproducibility, the eluates were analyzed by LightCycler RT-PCR targeting Cyclophilin A transcript (see table). Furthermore the reproducibility was shown by gel electrophoresis of the eluates. Reproducibility based on Cyc. A LC analysis
Sample Culture Cells 105 K562 cells 105 Hela cells 22.0 20.8 2.7 2.8 30 30 CP (mean) %CV n

CV: Coefficient of variance reflecting the reproducibility of the combination of MagNA Pure LC RNA isolation and LightCycler RT-PCR. CP: Crossing point

Volume ranges set by the MagNA Pure LC Software


Sample Volume * Elution Volume 200 l (containing 103-106 cels) 100 l
RT-PCR analysis targeting Cyclophilin A of total RNA purified from 105 K562 cells on the LightCycler Instrument

* Smaller sample volumes need to be filled up with an appropriate buffer to result in at least 200 l.

RT-PCR analysis targeting Cyclophilin A of total RNA purified from 105 K562 cells on the LightCycler Instrument

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55

Scalability To show the scalability of total RNA isolation, different amounts K562 cells (1 x 105, 2.5 x 105, 5 x 105, 1 x 106 cells) were processed on the MagNA Pure LC Instrument. The amount of RNA was determined by OD260 measurement.

To show the correlation between quantification by PCR (CPs) and OD260 measurement, the RNA was analyzed by LightCycler RT-PCR targeting Cyclophilin A transcript.

RT-PCR with theoretical 100% efficiency results in a difference of crossing points by 3.3, when the amplified amount of RNA increases by factor 10. RNA, isolated with the MagNA Pure LC Instrument, results in a difference of 2.9 crossing points, when analyzed by LightCycler RT-PCR.

MagNA Pure LC System Isolates High Quality Total RNA RNA was isolated from different cell lines (10 6 cells each) with the MagNA Pure LC Instrument. Purified RNA was analyzed by gel electrophoresis. The MagNA Pure LC isolated RNA proved to be excellent with respect to integrity.

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6.4

mRNA Isolation Kit I


Pack Size 1 Kit (192 reactions)

Cat. No. 3 004 015

Reproducibility based on Cyc. A LC analysis


Sample Blood whole blood (50 l) 24.3 21.5 21.3 1.2 2.3 1.2 30 30 30 CP
(mean)

%CV

Typical Performance Data


Sample Yield CP Sample Yield CP

PBMCs from 600 l blood WBCs from 200 l blood Culture Cells 105 K562 cells 105 Hela cells

Whole blood 10 l 25 l 50 l 100 l 0.6 ng 1.5 ng 3.5 ng 5.7 ng 26.9 25.6 24.2 23.5

Cultured Cells K562 1 x 103 1 x 104 1 x 105 1 x 106 0.7 ng 10.7 ng 145.4 ng 662.7 ng 26.5 21.9 17.5 15.0

17.9 15.7

1.3 1.5

30 30

PBMCs (isolated from l blood) 150 l 300 l 600 l 1200 l 10.1 ng 20.4 ng 43.7 ng 77.0 ng 22.1 21.1 19.9 19.4

Cultured Cells HeLa 1 x 103 1 x 104 1x 105 1 x 106 0.5 ng 19.3 ng 360.0 ng 1.8 g 25.7 20.6 16.3 13.9

CV: Coefficient of variance reflecting the reproducibility of the combinati on of MagNA Pure LC mRNA isolation and LightCycler RT-PCR. CP: Crossing point

WBCs (isolated from l blood) 100 l 200 l 400 l 600 l 5.8 ng 7.2 ng 14.7 ng 18.1 ng 22.1 21.1 19.9 19.4

Note: Yields were calculated via crossing points after RT-

PCR analysis using the Cyclophilin A as target transcript and the LightCycler Instrument. For calculations of sample mRNA concentrations a standard mRNA, purified from K562 cells, was used. Yields may differ depending on the origin of the sample material and the standard being used.
Volume ranges set by the MagNA Pure LC Software
Sample Volume Cells: 300 l (containing 103-106 cells) Whole Blood: 10-100 l WBCs/PBMCs: 300 l (containing WBCs or PBMCs) Elution Volume 25-100 l

LightCycler RT-PCR analysis of mRNA isolated from 105 HeLA cells with the MagNA Pure LC mRNA Isolation Kit I.

Reproducibility Different sample materials were processed with the MagNA Pure LC Instrument in replicates of 30. To demonstrate MagNA Pure LCs reproducibility, the eluates were analyzed by LightCycler RT-PCR targeting Cyclophilin A transcript (see table).

LightCycler RT-PCR analysis of mRNA isolated from WBCs with the MagNA Pure LC mRNA Isolation Kit I.

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Scalability To show the scalability of mRNA isolation sample materials whole blood (10 l, 25 l, 50 l, 100 l) PBMCs isolated from 150 l, 300 l, 600 l, 1200 l blood WBCs isolated from 100 l, 200 l, 400 l, 600 l blood culture cells HeLa and K562 (1 x 103, 1 x 104, 1 x105, 1 x 106 cells) were processed on the MagNA Pure LC Instrument. The eluates were analyzed by LightCycler RT-PCR targeting Cyclophilin A transcript.

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6.5

Total Nucleic Acid Isolation Kit


Pack Size 1 Kit (192 reactions)

Cat. No. 3 038 505

Performance Comparison Total nucleic acid from serum samples was isolated with the MagNA Pure LC Instrument and in parallel by a conventional spin column method. Eluted nucleic acid was analyzed with quantitative LightCycler PCR specific for Parvo Virus B19 DNA. Both methods show equal results (see graph).

Reproducibility Different sample materials containing 3.5 x 107 copies/ml of Hepatitis A Virus were processed with the MagNA Pure LC Instrument in replicates of 30. To demonstrate MagNA Pure LCs reproducibility, the eluates were analyzed by LightCycler RT-PCR (see table and graph).

Reproducibility based on LC analysis targeting Hepatitis A Virus


Sample (50 l volume) Plasma (citrate) Serum Whole blood (EDTA) (200 l volume) Plasma (citrate) Serum Whole blood (EDTA) 25.1 25.0 26.8 1.4 0.6 2.0 26.1 26.5 27.2 1.8 2.0 2.6 CP
(mean)

%CV

CV: Coefficient of variance reflecting the reproducibility of the combination of MagNA Pure LC and the LightCycler CP: Crossing point

Volume ranges set by the MagNA Pure LC Software


Sample Volume * Elution Volume 50 - 200 l 100 l

* Smaller sample volumes need to be filled up with an appropriate buffer (i.e. PBS) to result at least 50 l.

MagNA Pure LC isolated total nucleic acid from 200 l citrate plasma samples; RT-PCR analysis of Hepatitis A Virus RNA on the LightCycler PCR System.

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Scalability To show the scalability of total nucleic acid isolation, samples of plasma and serum containig 104 - 108 copies/ ml Hepatitis A Virus or Parvo Virus B19 were used. All samples

were processed on the MagNA Pure LC. The eluates were analyzed by LightCycler RT-PCR or PCR.

6.6

Cross contamination

In order to prove that there is no risk of cross contamination, a MagNA Pure LC Sample Cartridge was loaded with blood (for performing the DNA Isolation Protocol) or blood plasma (for performing the Total Nucleic Acid Isolation protocol), respectively. Half of the wells were spiked

with high amounts of plasmid DNA (5 x 1011 copies/ml) containing a specific sequence of Parvo B19 virus. Spiked and unspiked samples were loaded into the sample cartridge in a chequer-board pattern.

Blood

Blood + Parvo B19 DNA (5 x1011 copies/ml)

The eluates were tested for Parvovirus-specific DNA using a sensitive LightCycler PCR system, which was proven to detect less than 10 copies.
No cross-contamination was detected in the negative controls (16 per run).

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LightCycler PCR analysis of nucleic acids isolated with the MagNA Pure LC Total Nucleic Acid Kit from 16 positive and 16 negative samples (corresponding to the spiked and non spiked cartridge wells) resulted in two clearly distinguished bundles of curves.

LC-PCR analysis: No cross contamination of Parvo Virus B19 negative samples.

Electrophoresis of PCR products: Parvo-specific PCR products appeared in all lanes of spiked samples but in none of the non-spiked samples.

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7.
7.1

Specification
Typical Instrument Specifications
Instrument Type Sample Capacity Liquid Dispensing Capacity Dispensing Accuracy User Interface Power Source Power Consumption Dimensions Weight Benchtop stand-alone instrument Up to 32 tests per batch*) 51000 l 5100 l: < 3% CV 1001000 l: < 2% CV User-friendly interface, based on Windows 2000 operating system 100240 V AC (10%); 800 VA; 5060 Hz 800 VA max. 1000 mm (W) x 650 mm (D) x 890 mm (H) 151 Kg

*)With the 8-nozzle multipipet, 32 (4 x 8) samples can be processed at the same time. However, the number of samples in a single run can vary from 1 to 32.

7.2

Specifications of the HEPA filter

Filter Frame Treatment of frame Seal material Gasket Maximum temperature Maximum humidity Certificate Particle Characteristics

Glass fiber Aluminium Anodized and painted Self-digestive seal Neoplain sponge 800 C 100% relative humidity no > 0.3 m are 99.97% stopped

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7.3

Required Specifications for PC, Monitor and Printer

In order to avoid instrument failures, it is obligatory to use a standard PC, the Hewlett Packard Vectra VL420/1.7GHz. Experience has shown that the use of other computers can cause problems, especially if some software is pre-installed. The Hewlett Packard Vectra VL420/1.7GHz has been thoroughly tested together with the MagNA Pure LC Instrument and proved to be compatible with the MagNA Pure LC Software.

See the following table for detailed specifications of the recommended PC system: Note: Computer workstation configuration may vary from country to country due to component availibility and technical advances. To confirm exact computer configuration, please contact your local Roche Applied Science representative.

Recommended standard PC for MagNA Pure LC Instrument HP Vectra VL420/1.7GHz


Processor / CPU Cache Memory Hard disk drive CD device Floppy disk drive Graphics Ports Expansion slots Keyboard Mouse Operating System Software Warranty Intel Pentium IV 1.7 GHz Processor 256 KB 256 MB non-ECC 133 MHz DRAM Min. 20 GB UATA / 66 5.400 RPM HDD 48x IDE CD-ROM drive 3.5 1.44 MB FDD Matrox MAG G400 AGP graphics card, 2 x 8 MB SDRAM 1 serial ports, 1 parallel port, 4 USB ports 3 x PCI PS2 keyboard PS2 mouse Microsoft Windows 2000 Professional (english version) No software (e.g., virus scan software) should be pre-installed on the computer. 3 years local warranty

Monitor Printer

17"; 100 Hz; dot pitch, 0.28 mm min. HP 895 Cxi

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8.

Instrument Installation
the necessary facilities as detailed below. In case you want to move the MagNA Pure LC system to a different location contact your local Roche representative.

Installation of the MagNA Pure LC workstation is performed by a Roche Diagnostics service engineer only. Furthermore, the accompanying PC system comes with a preinstalled software. The user is responsible for providing See the following table for a list of installation requirements:
To ensure that the MagNA Pure LC workstation operates correctly, the installation site for the instrument should meet all these prerequisites and conditions: Location Power Source

Independent, grounded electrical power outlet which can supply 90 - 240 V AC, at 10 A and 50/60 Hz. Note: Because normal line voltage may fluctuate, we recommend the use of an Uninterruptible Power Supply (UPS). Caution: Do not plug any other electrical device into the outlet used for the MagNA Pure LC Instrument. Ensure the instruments power plug is always earthed. The instrument is intended for indoor use only. Place the instrument on a flat, stable, and vibration free surface. Do not place the instrument near any instrument or equipment that is likely to produce electrical noise and/or voltage fluctuation that will interfere with instrument operation. Do not place the instrument in direct sunlight or under a bright ceiling lamp. Light intensitiy inside the laboratory should not be higher than 2.5 kLux. If a window has screens ensure that no stripes of light can fall onto the region of the Reagent/Sample Stage, because this could activate the clot detection sensor. If you choose the Teleservice Support option from Roche Diagnostics, locate the instrument near a telephone connection.

Ceiling height Doors, elevators, corridors Table size required

At least 315 cm for unpacking the instrument. The instrument package is 115 cm (width) x 80 cm (depth) x 125 cm (height). 100 cm (width) x 70cm (depth) x 90 cm (height) plus additionally min. 100 cm at both sides of the instrument to open the side doors plus min. 90 cm above the instrument to open the front door. There should be enough space at the left side of the instrument to place the PC, because the right side is blocked due to the heat exhaustion.

Bench loading capacity Additional information

151 kg instrument plus 50 kg PC = approx. 200 kg It is to be noted that the MagNA Pure LC generates up to 700 Watt which are converted into heat. It is possible to cool the MagNA Pure LC with an appropriate cooling device. Adequate arrangements should be made for the free flow of air for effective cooling. The noise level of the instrument meets international specifications: < 62 dB

Altitude Room Temperature Relative Humidity Transient Category* Pollution Degree

up to 2000 m 1530C 80% or less (for temperatures up to 30C) II (*according to Installation categories) 2

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9.

Warnings and Cautions


Carefully read and understand the Roche Diagnostics safety statements contained in this section. This information should be kept for future reference and made available to new employees. Always keep this book available near the MagNA Pure LC Instrument for use by all operators.

In an emergency, immediately turn the power switch off and unplug the instrument.

This instrument is an electromechanical device that could cause electrical shock or injury if not operated according to procedures in this manual. Failure to comply with the instructions in this manual will void the manufacturers warranty and may expose the user to danger.

9.1

Important Safeguards

Do not let water or chemicals come into contact with any part of the equipment. Water or chemicals may cause damage and will void your warranty.

Do not touch the surface of the Heating Block. The block is hot enough (up to 90C) to cause an immediate burn.

Do not insert hands or fingers into either of the holes provided for discarding used tips and waste liquids (located near the front of the Reagent/Sample Stage). If the reagent-dispensing Nozzle Head moves suddenly, it may strike and injure your hand or fingers.

Unplug the equipment if it will not be used for extended periods of time.

When using the equipment, follow generally accepted procedures for quality control and method development. Do not attempt to disassemble the equipment. Service personnel trained by the manufacturer must perform repairs. The equipment contains no user serviceable parts.

Do not modify any part of the equipment. Modifications may cause a fire or malfunction; they may also void the manufacturers warranty.
Do not change or edit the Purification Protocols available in the MagNA Pure LC Software. This will lead to a malfunction of the MagNA Pure LC System!

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65

9.2

Precautions to prevent Contamination and Infection

The MagNA Pure LC Instrument was designed to ensure safe operation. However, since human sample material may be processed in the instrument, there is a possible risk of infection. Therefore, when handling potentially infectious material, always follow standard safety procedures and take the following steps into account to minimize the chance of contamination or infection: Always wear gloves and a mask when handling specimens and reagents. Keep the Tip Waste Slide clean. Do not operate the instrument without a MagNA Pure LC Waste Bag secured to the end of the slide. Discard the Waste Bag and the filled Liquid Waste Bottle (only necessary when performing the optional Liquid Waste Discard) after preparing each full set of samples (32 samples). After sample preparation is complete: Remove and autoclave all disposable plastics, if you worked with infectious sample material. Wipe the Reagent/Sample Stage with bleach (10% solution of sodiumhypochloride) and let it react for 10 min. To remove the bleach completely, wipe the instrument surface with 70% ethanol, followed by pure water. Note: After using bleach, ventilate the instrument for at least 1 hour. Accessories (e.g., Cooling Blocks, Reagent Tub Rack) may be treated the same way. Important: Never clean accessories in a dishwasher ! Wipe the front surface of the magnetic plate with 70% ethanol using a smooth cloth.

After cleaning the Reagent/Sample Stage with bleach and water/ethanol, decontaminate the instrument using the built-in UV-lamp, as follows (see Chapter B, section 9.2 for details): Close the door and click the Decontamination button. Set the decontamination time (recommended setting, 8 hours). Click the Start button. Note: Bleach can be used for disinfection and removal of contaminating nucleic acids and nucleases at the same time. For sole disinfection purposes one may also use commercially available disinfection reagent (please contact respective distributors in your country for details). For removal of contaminating nucleic acids and/or nucleases decontaminating agents like LTK008 or DNA/RNA ZAP may be used. Be aware that decontamination agents do not disinfect. Please follow the instructions of the respective manufacturer (see Chapter B, section 2.3.1 for details on decontamination agents). Important: The MagNA Pure LC Instrument is not a fully airtight device. There is an air flow under the instrument platform. Although total air flow from the platform is filtered through the HEPA filter (see Chapter A, section 2.3 for details), it cannot be ruled out completely that the platform atmosphere never leaves the instrument without prior filtration by the HEPA filter. Therefore, the MagNA Pure LC Instrument does not have the same functionality as a laminar flow biological safety cabinet and should not be used for analysis of infectious sample material unless additional safety measures ensure safe sample handling.

9.3

Position and Meaning of Warning Labels

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Symbol

Location

Meaning

on Block

Heating

CAUTION: HIGH TEMPERATURE When the MagNA Pure LC Instrument is turned on, the temperature of the Heating Unit automatically rises to 60C. During a run, it may rise to 90C!

on reagentdispensing Nozzle Head

KEEP HANDS AWAY While the instrument is operating, the reagent-dispensing Nozzle Head may move suddenly and hit your hands, causing injury!

near Tip Waste Disposal Slide

BIOHAZARD The Tip Waste Disposal Slide and Liquid Waste Bottle may become contaminated with biohazardous materials. Do not touch these areas without gloves or other biohazard protection gear!

on door of instrument

CAUTION
Do not install or run the MagNA Pure LC Instrument in direct sunlight! Do not run the MagNA Pure LC Instrument at room temperatures higher than 30C ! Ensure the 2 spacers are installed in their holder notches on the back of the

MagNA Pure LC Instrument ! EXPLOSIVE ATMOSPHERE


Be sure covers are mounted onto each filled reagent tub! If the fans are not running, do not leave reagents inside the instrument for longer

than 1 hour!
If MagNA Pure LC Instrument stops (e.g., due to power being turned off, instru-

ment being in STANDBY mode, power failure, or operating error), remove all reagents, then leave the instrument door open for at least 30 minutes!
If you alter the batch instructions (e.g., batches, reagents or volumes) or use new

ones, always calculate the sample alcohol content and perform an alcohol evaporating test!

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10. Marks of Conformity


The MagNA Pure LC Instrument has been manufactured according to EN 61010-1 (Safety regulations for Measuring, Control and Laboratory Instruments; Part 1: General Requirements [IEC 1010-1 + A1: 1992, modified]) and has been checked in accordance with all relevant safety standards prior to leaving the factory. The instrument has been approved for use by recognized testing institutions. This is confirmed by the following test/conformity symbols:

Acronym

Test Symbol

Testing Institution

GS

Certified by TV Product Service

CE

The instrument meets the protection requirements laid down in the European Concil directive 89/336/EEC on electromagnetic compatibility.

UL

Certified by Underwriters Laboratories Inc.

CUL

Certified by Underwriters Laboratories for Canada a testing facility recognized by the Standards Council of Canada (SSC)

Equipment to be connected must fulfill the standards set by IEC 950 (Information security in technical equipment, including electronic business machines).

Classification The MagNA Pure LC Instrument is classified as: ISM instrument (Industrial Scientific Medical Device), medium-sized, for industrial, laboratory and domestic use. Designed for stationary operation. Intended for worldwide use. Intended for evaluating pre-processed biological material.

Note on Use with Infectious Material The instrument should not be used to analyze infectious materials unless additional safety measures to ensure safe sample handling (e.g., placing the instrument in a laminar flow biological safety cabinet, or using a purification protocol which allows external sample lysis) are taken beforehand.

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11. Third Party Software


Additional software applications (third party software) should not be installed on the MagNA Pure LC computer. Such software may cause the MagNA Pure LC software to malfunction. If other software packages are installed, reliable operation of the system can no longer be guaranteed. Install other software packages only at your own risk.

12. Revision History


Software Version Manual Version Date of Publication

1.0 2.0 2.1 3.0

1.0 2.0 2.1 3.0

December 1999 June 2000 May 2001 August 2002

This Operators Manual is published by Roche Diagnostics GmbH, a division of F. Hoffmann-La-Roche Ltd, CH-4070 Basel, Switzerland. Questions or comments regarding the content of this manual can be directed to the following address: Roche Diagnostics GmbH Roche Applied Science Nonnenwald 2 D-82372 Penzberg Attention: Support New Systems, Department BP-7 Fax No. +49 8856 60 6937 Alternatively you may contact your local Roche Applied Science representative or visit our Internet homepage,
http://www.roche-applied-science.com Note: Please have the serial number of your instrument

MagNA Pure is a trademark of a member of the Roche Group. Windows and Windows 2000 are trademarks of Microsoft Corporation in the USA and other countries. LightCycler is a trademark of a member of the Roche group. The technology used for the LightCycler system is licensed from Idaho Technology Inc., Salt Lake City, UT, USA. AMPLICOR and COBAS AMPLICOR are registered trademarks of Roche Molecular Systems, Inc. licensed to Roche Diagnostic, Inc. DNA/RNA ZAP is a trademark of Ambion, Inc. LTK-008 is a trademark of Biodelta Kohrsolin is a registered trademark of Bode Chemie Hamburg MessageSender is a trademark of JTECH Communications, Inc., Boca Raton, FL, USA.

available when contacting us. Every effort has been made to ensure that all information contained in this Operators Manual is correct at time of printing. However, Roche Diagnostics reserves the right to make any changes without notice as we periodically improve the instrument. 2000/2001/2002, F. Hoffmann-La-Roche Ltd All rights reserved.

13. License
The license for MS Windows 2000 has been registered at Microsoft by Roche Diagnostics. The license is the property of the owner of the MagNA Pure LC Instrument. The purchase of MagNA Pure LC reagent kits does not convey any licenses or other rights for the performance of PCR.

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69

14. Warranty
For warranty conditions, refer to the MagNA Pure LC purchase agreement. Contact your local Roche Diagnostics representative for further information

15. Technical Support


www.roche-applied-science.com
to order, solve technical queries, find product information, or contact your local sales representative.

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Chapter B

How to Operate the MagNA Pure LC System

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1.

Overview over the Workflow


them into Reagent Tubs, which are placed on the instrument Reagent/Sample Stage. Pre-programmed purification protocols containing the instructions for all isolation steps are pre-installed together with the software. The user selects an appropriate purification protocol depending on the kind of sample material and the type of nucleic acid that is to be isolated. The user then provides the software with details of the samples, fills the Reagent/Sample Stage with the required reagents and disposable plastics, and finally starts and monitors the run. This section of the manual gives you detailed instructions on performing these steps. See the following flow chart for an overview over the main handling steps required for preparation and execution of a purification run:

The MagNA Pure LC Instrument automatically performs a nucleic acid purification starting from crude sample material, with a final end-product of high-pure isolated nucleic acid ready for downstream applications such as PCR. The instrument automatically performs all steps of the purification procedure with specially designed nuclease-free, disposable Reaction Tips. These Reaction Tips not only transfer the lysed sample material, but also serve as reaction vials for the isolation procedure. Within the tips, nucleic acids are bound to magnetic particles (MGPs or SMPs; see Chapter B, section 5 for details), washed free from debris and impurities, and finally eluted from the magnetic particles in pure form. Eluted nucleic acids are then transferred into the wells of a Storage Cartridge, ready for further use or storage. All reagents needed for the purification process are included in the MagNA Pure LC nucleic acid isolation kits. The reagents are supplied for the purification process by filling

System Setup

Switch on the instrument and the computer Log-In and start the software Place the Waste Bag and (optionally) the Waste Bottle Place or change the Liquid Drop Catcher disposable

Sample Ordering

Place the filled Reagent Tub Rack and all other required disposable plastics on the Reagent/Sample Stage Place samples into the Sample Cartridge on the Reagent/Sample Stage Confirm correct preparation of the Reagent/Sample Stage. If the Start Information Screen was opened from the A-Ring Ordering Screen enter the Sample Cartridge barcode.

Open the Sample Ordering Screen. In case, you work with MagNA Pure LC Cooling Block, A-Ring in combination with COBAS AMPLICOR tests, open the A-Ring Ordering Screen. Select the appropriate purification protocol Specify protocol data Specify sample data. On the A-Ring Ordering Screen enter sample barcodes.

Batch run

Start the purification run and monitor it on the Batch Status Screen Evaluate the outcome of the purification run on the Result Screen

Post Elution run

Preparation for Operation


Open the Start Information Screen Fill the required isolation reagents into the appropriate Reagent Tubs and place them in the Reagent Tub Rack (outside the instrument)

Open the Post Elution Steps Screen and create or open a Post Elution Protocol Perform automatic PCR setup using the Post Elution function

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73

2.
2.1

System Setup
Switching on the Instrument

The main switch of the instrument is located at the lower left side panel of the instrument. See Chapter A, section 2 for a detailed description of the instrument parts.
Note: The instrument needs max. 20 min from power

After switching on the instrument, 4 LEDs at the lower right of the instruments front panel indicate the current status of the MagNA Pure LC Instrument:

switched on to warm-up.

Green: Power-On Yellow: Standby Orange: Batch-Running Red: Error

MagNA Pure LC status indication by front side LEDs

LED : Power-On : Standby

Description Power supply is adequate and all MagNA Pure LC functions work correctly. When the MagNA Pure LC Instrument is inactive for several minutes, the robotic arm returns to Home position and power is not supplied to the motors. When a command is sent, the instrument automatically leaves Standby Mode and returns to Power On Mode.

Important: Ensure that no obstacle is present on the instrument Reagent/Sample Stage. This may lead to damage, when the robotic arm moves back to the Home position.

Note: In Standby Mode, power is still supplied to the Heating and Cooling Units. : Batch Running During a nucleic acid preparation run, the Batch Running LED remains on. The door is locked and cannot be opened. : Error If an error occurs during a purification or a Post Elution run, the "Error-LED" lights up. Even after the cause of the error has been eliminated and the protocol has been restarted, this LED will remain switched on. To turn the LED off, you must exit the software.

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2.2

Log-in and Start Up Procedure for MagNA Pure LC Software

Access to the software is password restricted. Only users with authorized user names and passwords have access to the software. The log-in procedure is done via the Windows 2000 logon function during start of the operating system.

MagNA Pure LC Standard Users, who can perform purification and Post Elution runs, have to be registered by the User Administrator first, before they can access the software.

The different access levels and privileges of MagNA Pure LC users are:
Access levels and priviliges
User User Administrator Username admin Priviliges
controls software access and priviliges of

all Standard Users


registers the unique user name for each

new Standard User


installs new blocks and protocols

(see Chapter B, section 9.4 and Chapter B, section 9.5). Standard User
user specific defined by User can set up and run a nucleic acid

preparation by selecting an existing protocol from a menu.


can perform a Post Elution run

Administrator

2.2.1 Log-In as User Administrator Before you Begin Make sure that your MagNA Pure LC Instrument is connected correctly to the PC system and that the instrument is switched on.

Start the MagNA Pure LC software by following the steps described below:

Turn on the computer to start Windows 2000 When the Begin Logon message appears press the

The MagNA Pure LC software starts automatically.

keys <Ctrl>, <Alt>, and <Del>. In the Windows 2000 logon registration window type in the user name ADMIN. Enter your password. Note: During installation of the system, the person designated to be the User Administrator should choose a personal password. Confirm by pressing OK.

After an informative screen telling you that the program files are loading you will see either the User Administrators or the Standard Users Main Menu Screen.

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75

User Administrators Main Menu Screen


Note: Which functions are available on the Main Menu Screen depends on the privileges of the user being logged-in.

With MagNA Pure LC software 3.0 all necessary functions are accessible from the menu bar. Every software screen provides the same main menu points: File Edit Actions Help

While the functions of the File, Edit, and Actions menu differ from screen to screen, the Help menu is identical on every screen. The Help menu is described in detail in Chapter B, section 6.

Exit
Closes the Main Menu Screen and returns to the Windows 2000 desktop.

Leave Message
Allows creation of text messages for other MagNA Pure LC users. This function is described in detail on in Chapter B, section 9.3.

User Administration
Opens the User Administration Screen.

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Operations performed on the User Administration Screen

The User Administrator uses the functions of the User Administrator Screen to register or delete MagNA Pure LC Standard Users.

Adding a new user

To add a new user, select the entry 'Add New User'

Note: The Description field (defines the user level) always

from the 'Actions' menu:

reads "Standard User" and cannot be changed or edited. If a paging unit is installed to your MagNA Pure LC system and configured, you can add the identification numbers of the individual pagers into the Pager ID field. A user can then be automatically notified by the pager in case of an error or warning message, or when the purification run is finished. The text appearing in the messaging window will be displayed on the pager, too. Spelling of the User Name has to follow certain rules to guarantee reliable function of the operating system and the MagNA Pure LC software: A User Name must not be identical to any other user name or local user group name A User Name may consist of up to 20 characters (either small or capital letters) with no space in between Note: Instead of 'space' you may use '_', e.g., 'firstname_name' A User Name must not contain the following characters: " / \ [ ] : ; = , + * | ? < > SPACE A User Name must not consist solely of dots (.) The User Name defined with the Add New User function is used as user name for Windows 2000 logon, too. During the first Windows 2000 logon, the new user is prompted to define a new password.

The Add New User window will open:

To register a new user, type the new user name into

the User Name field (e.g., user_1).

Confirm by clicking the OK button.

The new user will appear in the User Name Table:

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Deleting a user account


To delete a user account first select the respective user A confirmation window appears asking you whether

entry in the User Name Table.

you want to delete the selected user account:

In the File menu choose the option 'Cut'.

Deleting a user password


To delete only the password of a user without deleting A confirmation window appears asking you whether

the users account select the respective user entry in the User Name Table.

you want to delete the password of the selected user account:

In the Actions menu choose the option 'Delete

Password':

2.2.2 Log-In as Standard User Before you Beginn Make sure that your MagNA Pure LC Instrument is connected correctly to the PC system and that the instrument is switched on.
Turn on the computer to start Windows 2000 When the Begin Logon message appears press the keys

Program Start Start the MagNA Pure LC software by following the steps as described below:

The MagNA Pure LC software will start automatically.

You will see the Standard Users Main Menu Screen. <Ctrl>, <Alt>, and <Del>. In the Windows 2000 logon registration window, type in your user name as specified by the User Administrator. Type in your password (if defined). Confirm by pressing OK.

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Standard Users Main Menu Screen

The Standard Users Main Menu Screen provides access to the main functions that are needed for setup of the instrument and performance of a purification run.
Note: In MagNA Pure LC software 3.0 all necessary func-

tions are accessible from the menu bar. Every software screen provides the same main menu points:

File Edit Actions Help While the functions of the File, Edit, and Actions menu differ from screen to screen, the Help menu is identical on every screen. The Help menu is described in detail in Chapter B, section 6.

Opens the Sample Ordering Screen for selecting the purification protocol, specifying protocol parameters, and entering sample information. This function is described in detail on in Chapter B, section 3. Opens the specially featured Sample Ordering Screen for users of the A-Ring Cooling Block for selecting the purification protocol, specifying protocol parameters, and entering sample information. A-Ring Ordering provides a strict user guidance (e.g., prompt for read in of sample barcodes) to ensure reliable sample tracking. In other respects its functionality is identical to the conventional Sample Ordering Screen. This function is described in detail in Chapter B, section 8. Opens the Post Elution Steps Screen to create a new Post Elution Protocol or to modify an existing Post Elution Protocol, and to start the Post Elution procedure. Displays the Decontamination Screen. The UV lamp used for decontamination of the working area of the instrument is programmed by specifying the decontamination time. This function is explained in detail in Chapter B, section 9.2.

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Allows creation of text messages for other MagNA Pure LC users. This function is described in detail in Chapter B, section 9.3. Opens the Operators Manual PDF-file. Closes the Main Menu Screen and returns to the Windows 2000 desktop. Locks or unlocks the instrument door. Depending on the current status of the cover the button may read Lock or Unlock Door.

Note: For safety reasons, you cannot operate the MagNA Pure
LC Instrument unless the door is closed. The door can only be opened when:
Main power is off, the instrument is in Standby Mode, or you click the Lock/Unlock Cover button on the

MagNA Pure LC Main Menu Screen The door has an electromagnetic and mechanical lock, which prevents the door from being opened while the robotic arm is moving. In addition to the electromagnetic door lock (controlled by the Lock/Unlock Cover button), the instrument has two independent Door Open/Door Closed sensors that ensure the robotic arm does not move while the door is open. Click this button only when you need to raise the door and change components on the Reagent/Sample Stage. Moves the robotic arm back to the home position, which is located at the right corner in the rear of the Reagent/Sample Stage. The button is used after exchange of the Liquid Drop Catcher, after Tip Discard or after the robotic arm has stopped somewhere on the Reagent/Sample Stage during a preparative run due to an error.

Important: Ensure that there are no obstacles on the Reagent/


Sample Stage, when activating this function. Ejects the Reaction Tips from the nozzle head into the Waste Bag. Use this button if the instrument has stopped during a purification run due to an error and Reaction Tips are still present on the nozzle head. Removes all liquid waste left after completion of a purification run within the Processing Cartridges into the Liquid Waste Bottle.

Important: No volume control for the Liquid Waste Discard


Bottle exists. Thus, when using this function the user must control the volume level in the Liquid Waste Discard Bottle before starting Liquid Waste Discard.

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Allows installation of the Liquid Drop Catcher disposable (which is identical to the Reagent Tub Lid Seal) into the Liquid Drop Catcher (see Chapter B, section 4.3 and Chapter B, section 2.4 for details). After clicking the button, the robotic arms moves to a position above the Processing Cartridge I tray and the nozzle head moves forward. The Liquid Drop Catcher is located on top of the magnetic plate. Moves the robotic arm to a position above the front Processing Cartridge positions. You have now access to the nozzle to exchange them. See Chapter C, section 2.2.2 for details. Status indicators for Heating Unit (Elution Cartridge), Cooling Unit 1 (Storage Cartridge) and Cooling Unit 2 (MagNA Pure LC Cooling Blocks for Post Elution). If the required temperature for operation of the instrument is not reached yet, the status indicators are red and display FAIL. If the correct temperature is reached they switch to green and display PASS. Operation of the instrument is only possible if all status indicators are on green.

Note: When the MagNA Pure LC software is opened, Heating


and Cooling units are automatically set at 60C and 7.5C, respectively.

2.3

Placing the Accessories on the MagNA Pure LC Reagent/Sample Stage

Before the disposable plastics needed for the isolation run can be placed on the Reagent/Sample Stage, the accessories that hold the disposables have to be installed. These are:

the accessories and decontaminate them before placing them back again on the Reagent/Sample Stage. Before placing the Reagent Tub Rack on the Reagent Sample Stage, it should be filled outside the instrument with Reagent Tubs containing the required isolation reagents Place the MagNA Pure LC Cooling Block(s) into Cooling Unit 2 of the Reagent/Sample Stage, if you want to perform a Post Elution Run directly after a purification run These accessories are described in detail in Chapter A, section 3.

The Reaction Tip Racks (for large and small Reaction Tips) The Tip Waste Disposal Slide The Liquid Waste Funnel and the Liquid Waste Funnel Tray

Note: These accessories may remain on the Reagent/Sample

Stage during the whole operation time of the instrument, as long as no contamination is assumed. In that case remove

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2.3.1 Decontamination of the Workstation

At the end of each working day on the MagNA Pure LC workstation, the instrument should be decontaminated from nucleic acids using the built-in UV lamp (see Chapter B, section 9.2 for details of use). For safety reasons it is recommended to decontaminate the surface of the workstation and the accessories additionally by wiping it with decontaminating reagents that destroy

any nucleic acids or DNases and RNases. Also decontaminate the work area where the manual handling steps are performed. For decontamination either bleach (10% solution of sodiumhypochloride; see detailed instructions in Chapter A, section 9.2) or the following commercially availabe reagents may be used (follow the working procedures given by the respective manufacturer):

Decontaminating reagents

Reagent LTK-008

Description is a ready to use reagent, especially designed to remove contamination through DNA, RNA, DNAse, RNAse, bacteria and phages from work surfaces.

Manufacturer biodelta Wielandstr. 28a 32545 Bad Oynhausen Germany Phone: +49-5731-792-110 Fax: +49-5731-792-333 Suppliers: USA: www.tetra-link.com CAN: www.gordontech.com

Order Number 200-000

DNAZap

completely degrades contaminating DNA and RNA at the level of PCR sensitivity

Ambion Inc. 2130 Woodward Austin, Texas, USA http://www.ambion.com/ catalog/CatNum.php?9890

9890

RNase Zap

Completely removes RNase contamination

Ambion Inc. 2130 Woodward Austin, Texas, USA http://www.ambion.com/ catalog/CatNum.php?9780

9780 9782 9784

Important: Do not spray any reagent directly on the instru-

ment or accessory surfaces or into the machine. Electronic and optical devices of the machine might be damaged. Only decontaminate by wiping. Never clean the MagNA Pure LC Cooling Blocks used for Post Elution in a dishwasher. If infectious sample material is used, additional safety measures have to be taken by using desinfection reagents.

The above mentioned decontaminating reagents are not always sufficient to disinfect the surface of the instrument or accessories. Gloves must be worn during any handling of disposable plastics, all manipulations inside the workstation and for all manually performed pipetting steps.

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2.3.2 Use of the Dispo Lockbar

The Dispo Lockbar (figure 1 below) holds the disposable plastics of the Prologue unit (Sample Cartridge, Reagent Tubs, Reaction Tip Trays) in their correct positions on the Reagent/Sample Stage. The Lockbar must be raised before you can position accessories (Reagent Tub Rack, Reaction Tip Tray Rack) or

disposables (Sample Cartridge, Reagent Tubs, Reaction Tip Trays) on the Reagent/Sample Stage, to avoid pulling them out of their location during a pipetting step. A purification run (or a movement of the robotic arm) can only be started after the Dispo Lockbar has been closed.

To raise the Lockbar, locate the handle at the right front of the bar...

... flip it up

and raise the bar out of the way.

After you have placed disposables on the Reagent/Sample Stage, reverse the above steps to lower and lock the Dispo Lockbar in place.

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2.3.3 Positioning the Reagent Tub Rack and the Reaction Tip Racks

Place the Reagent Tub Rack into position 2 of the Reagent/ Sample Stage (as shown in the photo below). Place Reaction Tip Rack L (large) into Reagent/Sample Stage position 3 and Reaction Tip Rack S (small) into Reagent/Sample Stage position 4 (refer to Chapter A, section 2.4 for the numbering of Reagent/Sample Stage positions).
Notes:

It is recommended to fill the Reagent Tubs with the appropriate reagents outside the instrument. After filling the reagent close the tub with a Reagent Tub Lid, and then place the filled Reagent Tub in the Reagent Tub Rack. After placing the Reagent Tubs put the Reagent Tub Rack in its position on the Reagent/Sample Stage. Handling of Reagent Tubs is explained in detail in Chapter B, section 4.2.5.
Important: Magnetic particles should be added as the last

The Reagent Tub Rack must be placed correctly. There is a positioning pin on the Reagent/Sample Stage to prevent incorrect placement of the rack: The cut corner of the rack hast to point to the front right side. Reaction Tip Rack L (large) does not fit in the front position, but Reaction Tip Rack S (small) will fit in the rear position. Do not inadvertently place Reaction Tip Rack S in the wrong position.

reagent shortly before the start of the isolation run, because they tend to sediment fast, and to avoid alcohol evaporation.

Reaction Tip Racks Reagent Tub Rack Rack L Rack S

Stage Layout

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2.3.4 Positioning the Liquid Waste Funnel and Tip Waste Disposal Slide Funnel: Place the Liquid Waste Funnel in the compartment at the front left of the Reagent/Sample Stage (position 11), as indicated in the photos below. Always insert the tip of the Liquid Waste Funnel into a disposable Liquid Waste Bottle, as shown. Important: If using the optional Liquid Waste Discard function (see Chapter B, section 3 and Chapter B, section 9.1) always check the liquid volume inside the Liquid Waste

Bottle. The MagNA Pure LC Instrument does not automatically check the liquid level inside the waste bottle. If the bottle is already filled activation of Liquid Waste Discard will lead to overflow of the bottle and possible contamination of instrument or laboratory environment. Do not autoclave or wash the Liquid Waste Funnel at a temperature higher than 60C.

Liquid Waste Funnel

Tip Disposal Hole

Slide: Insert Tip Waste Disposal Slide into the slot provided in front panel. Hook the slide into the notches.
Tip Waste Slide

Magnet

To ensure reliable tip disposal, always hook the slide into the notches correctly. If the waste slide is inserted correctly the circular mark for placing the magnet on the slide can be seen on the lower end of the top side of the slide. The magnet holds a disposable MagNA Pure LC Waste Bag on the slide.

Note: After a certain time of use deposits may be forming

on the inner side of the Tip Waste Disposal Slide. This is due to residues of reagents, magnetic beads, and samples at the reaction tips which are discarded through the Waste Tip Slide into the Waste Bag. Because these deposits may contain infectious material the Waste Tip Slide should be disinfected. An overnight treatment of the Waste Tip Slide with e.g., 1.5% Kohrsolin solution followed by cleaning in a dish washer is recommended.

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2.3.5 Positioning MagNA Pure LC Cooling Blocks

The MagNA Pure LC Cooling Blocks are positioned in the large Cooling Unit 2, located in the right front quadrant (position 12, see Chapter A, section 2.4) of the Reagent/ Sample Stage. Into Cooling Unit 2 one or two of the small type MagNA Pure LC Cooling Blocks (LightCycler Centrifuge Adapters, 96-well PCR plate, Reaction Tubes) can be placed in any combination. In place of these pairs of small Cooling Blocks, Cooling Unit 2 can also hold one MagNA Pure LC Cooling Block for LightCycler Sample Carousel or COBAS Amplicor A-Rings, respectively. The tables below give examples of cooling block combinations that can be placed in Cooling Unit 2 at the same time. The MagNA Pure LC Cooling Blocks are not needed for the isolation run, but only for a Post Elution run, when setup of PCR reactions from samples and master mixes takes

place. See Chapter A, section 3 for an overview and description of the various available MagNA Pure LC Cooling Blocks. If a Post Elution run should be performed after the isolation run, MagNA Pure LC Cooling Blocks may be placed in advance. Another option is to place the cooling blocks at 4C-8C in a refrigerator, and to put them in Cooling Unit 2 shortly before the start of the Post Elution run.
Important: Do not place MagNA Pure LC Cooling Blocks in

a deep freezer at -20C for fast cooling. This would impair propper fitting of the cooling block in the Cooling Unit 2. Furthermore, it would lead to immediate freezing of reagents placed in the wells of the cooling block and to pipetting of incorrect volumes.

Examples of cooling block combinations that can be placed in tub at the same time.

Left side of Cooling Unit 2 Cooling Block for: - 96-well PCR Plate - LightCycler Centrifuge Adapters - 96-well PCR Plate - Reaction Tubes *) - LightCycler Centrifuge Adapters - Reaction Tubes - 96-well PCR Plate - LightCycler Centrifuge Adapters - Reaction Tubes *) *)

Right side of Cooling Unit 2 Cooling Block for: - LightCycler Centrifuge Adapters - 96-well PCR Plate - Reaction Tubes *) - 96-well PCR Plate - Reaction Tubes *) - LightCycler Centrifuge Adapters - 96-well PCR Plate - LightCycler Centrifuge Adapters - Reaction Tubes *)

Entire Cooling Unit 2 Cooling Block for: - LightCycler Sample Carousel*) - COBAS AMPLICOR A-Rings

Note:
To position the LightCycler Carousel correctly into the Cooling Block, ensure the pin of the block fits correctly into the hole in the Carousel. Press firmly to ensure the Carousel is seated correctly!

The MagNA Pure LC Cooling Blocks for Reaction Tubes, LightCycler Sample Carousel, and for A-Rings are not included in the instrument package, but can be ordered separately. Please refer to the table in Chapter A, section 3.2.1 or see the Ordering Guide in the Appendix.

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2.4

Placing and Changing the Liquid Drop Catcher Disposable

The nozzle head located at the lower end of the robotic arm performs all pipetting steps. During the procedure, the nozzles hold the disposable Reaction Tips. Whenever the nozzle head moves over the Reagent/Sample Stage, the Liquid Drop Catcher, which is located on top of the magnetic plate, moves underneath the tips to prevent possible drops falling from the tips which could lead to contamination of the Reagent/Sample Stage, reagents or other samples. The Reagent Tub Lid Seal serves as an easily removable tray to hold potential drops. It is recommended to change the Tub Lid Seal in the Liquid Drop Catcher at least before the first purification run of a working day. It is also recommended to exchange the Liquid Drop Catcher disposable before every new RNA purification run, to avoid any possible RNase contamination.
To place the Lid Seal on the Liquid Drop Catcher, do the following:
Log onto the MagNA Pure LC Software and click the

Liquid Drop Catcher with tray. A Lid Seal serves as the disposable for the Liquid Drop Catcher

Start Program button on the Title Screen. On the Main Menu Screen click the button Change Dropcatcher:

Keep pushing until the left side of the Lid Seal reaches the end of the channel:

Left side of Lid Seal

Important: Ensure that no obstacles are present on the Reagent/Sample Stage.


The robotic arm moves to a position above Processing

End of Channel

Cartridge I and the nozzle head moves forward, which makes inserting the Lid Seal easier (see picture above).
Slide a Lid Seal (using your fingers or clean plastic

forceps) into channel of Liquid Drop Catcher from the right:

While you are sliding the Seal, it may ride upward, out of the channel. Once the left side of the Seal reaches the end of the channel, the Seal falls automatically into the correct position.
After you have placed the Lid Seal, close the instru-

ments door and click on the button Home:

After the robotic arm moves to the home position,

the instrument door will remain closed. To open the door, click on the button Unlock Door:

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To remove the Lid Seal from the Liquid Drop Catcher, do the following:
Log into the MagNA Pure LC Software and click the Continue pushing the Lid Seal from the left side with

Start Program button on the Title Screen. On the Main Menu Screen click the button Change Dropcatcher:

a large Reaction Tip or forceps, until you can reach it with your fingers:

Forceps or
The robotic arm moves to a position above Proces-

Large Tip

sing Cartridge I and the nozzle head moves forward, which makes removing the Lid Seal easier (see picture above).
With your finger or clean plastic forceps, start Grab the Lid Seal and remove it from the right side

pushing the Lid Seal from the left side of channel:

of the channel:

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3.

Sample Ordering
3.1 Opening the Sample Ordering Screen

Before starting a purification run the user has to provide the software with the necessary information on protocol and sample parameters. The most important and obligatory ones are: Purification protocol used Amount of sample and elution volume Number of samples Name of samples

Follow the instructions under "Log-in as Standard

User" Chapter B, section 2.2.


On the Main Menu Screen click on the button

"Sample Ordering":

Input of sample and protocol parameters is done on the


Sample Ordering Screen.

The MagNA Pure LC Sample Ordering Screen will

appear. Provided with this information the software calculates the type and amount of disposables and reagents needed, and displays this information on the Start Information Screen. The user can then place the required disposable plastics and reagents on the Reagent/Sample Stage.

The Sample Ordering Screen

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Menu Bar of the Sample Ordering Screen

All functions necessary for sample ordering are found in the screens menu bar:
File Menu
New Sample Order Clears the Sample Ordering Table and all other entry and data fields of the Sample Ordering Screen (after confirmation). Use this function if you want to prepare a new Sample Order. Load Sample Order Loads a previously saved Sample Order File (*.sod). Existing screen data will be lost (after confirmation). Save Sample Order Saves the sample ordering screen data as Sample Order File (*.sod) for re-use for subsequent purification runs. Print Sample Name Prints a list of the sample names corresponding to their order in the Sample Cartridge. This printout can be used as a loading scheme for the Sample Cartridge. Print Sample Order Prints the current sample order information. Use this function to produce a permanent record of your information. Print Cartridge Barcode Generates and prints a barcode label (if an optional barcode printer is installed) which can be used to track and identify the Sample and Storage Cartridge of the current experiment. The Cartridge Barcode is created by the software and consists of the last three digits of the instruments serial number plus a batch run number. The Cartridge Barcode is identical to the Batch Identification Number (BatchID) which is also listed on the Result Screen and Post Elution Result Screen printout. The barcode can subsequently be read-in for naming of the Sample Order and Result Screen file (if an optional Barcode Reader is installed). When you want to re-load the Sample Order or Result Screen file, simply read-in the barcode from the Storage Cartridge.

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This function is especially necessary for the A-Ring Ordering procedure. On a Start Information Screen initiated from an ARing Ordering Screen you are prompted to enter a barcode for the Sample Cartridge which you can create beforehand using the Print Cartridge Barcode function. Printer Setup Lets you choose an appropriate printer driver and printer settings. Close Closes the Sample Ordering Screen and returns to the Main Menu Screen.

Edit Menu
Cut Cuts and thereby removes a selected sample from the Sample Ordering Table and from the Sample Cartridge Graphic. The sample data is stored within the clipboard memory and can be used for pasting it into a different sample position. Copy Copies the sample data of a sample selected in the Sample Ordering Table into the clibboard memory. Paste Inserts previously copied or cut sample data into a sample position selected in the Sample Ordering Table.

Actions Menu
Open Result Screen Loads a previously saved purification result screen (*.ird). Existing screen data will be lost (after confirmation). Open Post Elution Opens the Post Elution-Steps Screen to create a new Post Elution Protocol or to modify an existing Post Elution Protocol and to start the Post Elution procedure.

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Entry Fields of the Sample Ordering Screen


After clicking on the down-arrow button, a pull-down menu appears which lists Purification Protocols installed into the software. Selected a purification protocol appropriate for the purification run. After selection the name of the selected purification protocol is displayed.

After clicking on the down-arrow button, a pull-down menu appears which lists existing Post Elution Protocols that have been defined and stored previously and can be selected for this run. The selected Post Elution Protocol will start directly after completion of the purification process, except an error has occurred during the batch run.
Note: To be selectable from the Post Elution Protocol menu Post Elution protocol files have to be stored within the directory '..\MagNAPure\Protocols\PostElution'. Important: Note that there will be no pausing between the purification run and the Post Elution run. Therefore, all disposable plastics and reagents needed for Post Elution must be placed on the Reagent/Sample Stage in advance. The number of Reaction Tips required for Post Elution pipetting cannot be displayed on the Start Information Screen. Only the Reaction Tips needed for the actual purification run are calculated. Thus, it is recommended to place a complete set of Reaction Tips if performing a Post Elution run in direct combination with a purification run. Entry of information into the following five entry fields is optional. Data will be stored within a saved Sample Order or Result Screen file for documentation purposes. In addition, it is transferred to the LightCycler SAM-file that can be created from the Post Elution Result Screen. Enter the lot number of the MagNA Pure LC Isolation Kit used. This lot number is stored within a saved Sample Order and Result Screen file for documentation purposes. In addition, it is transferred to the LightCycler SAM-file that can be created from the Result Screen. If you use nucleic acids isolated on the MagNA Pure LC Instrument as template for a subsequent LigthCycler PCR you may enter both the kit name and the lot number of the LightCycler generic reagent kit (e.g., LightCycler FastStart Master Hybridization Probes or LightCycler RNA Master) used here.

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If you use nucleic acids isolated on the MagNA Pure LC Instrument as template for a subsequent LigthCycler PCR you may here enter the lot number of the LightCycler parameter specific reagent kit or of the primer/probe set used, respectively. Enter lot-specific information on a control template (e.g., heterozygous plasmid control when performing a mutation detection) here. If you use nucleic acids isolated on the MagNA Pure LC Instrument as template for a subsequent LigthCycler PCR and you have several LightCycler sample carousels on hand you may enter the carousel number for identification purposes here. The carousel number is stored within a saved Sample Order or Result Screen file for documentation purposes. In addition, it is transferred to the LightCycler SAM-file that can be created from the Post Elution Result Screen. Type the desired sample volume in l into this field. Note: If you select a purification protocol that is based on an external pre-treatment of the sample (e.g., Total_NA External_Lysis) the entry field is designated Lysed Sample Volume. In this case you should not enter the volume of the primary sample but the volume of the complete lysate. Type the desired elution volume in l, into this field.

Type the desired dilution volume in l, into this field. During the Prologue phase of a purification protocol the Dilution Buffer, which is identical to Elution Buffer, is pre-pipetted from an additional Reagent Tub into the Storage Cartridge. Liquid Waste Discard check box: Programs the instrument to automatically discard all liquid waste at the end of a run. Important: No volume control for the Liquid Waste Discard Bottle exists. Therefore, when using this function you should always control the volume level within the waste bottle before starting the run. Note: With software version 3.0 it is also possible to perform a liquid waste discard as a separate function directly from the Standard User Main Menu Screen.

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Graphical Elements of the Sample Ordering Screen Sample Cartridge Graphic


Used for assigning a sample to a particular position in the Sample Cartridge during Sample Ordering.

Sample Order Table

Lists the samples and their data (name, position) in line blocks of 8 samples to ease orientation. In addition, it is used for direct entry of sample data. #: Displays the sample numbers Note: The software automatically assigns the sample numbers as the user enters each set of sample information. Sample Name: Displays the corresponding sample name. Enter sample names manually with the keyboard or automatically with a barcode reader. Comment: If desired, you may enter a brief descriptive comment for the sample. Pos: Displays the well coordinate of each sample within the Sample Cartridge.

Stage Setup Button

Opens the Start Information Screen on which correct placement of all required reagents and disposable plastics on the Reagent/Sample Stage is confirmed, and from which the purification process is actually started. Note: It is not possible to proceed to the Start Information Screen if not all sample and protocol data was put in correctly.

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3.2

Selecting the appropriate Purification Protocol

For every MagNA Pure LC reagent kit a specific purification protocol exists. The purification protocol contains the information about the reagents included in the kit, the order of pipetting steps, and the incubation times for the various reaction steps of the purification procedure. For some reagent kits more than one purification protocol may exist, if specific instructions for different sample materials or variant grades of purification are possible. Purification protocols are pre-installed together with the software itself. If new reagent kits are offered or additional protocols for further sample materials are available, new purification protocols can be installed using the Protocol Installer software module, which is explained in detail in Chapter B, section 9.5.

Besides the fixed purification run parameters that are provided by the purification protocol itself, the software needs additional, run specific parameters, that have to be provided by the user. These are:
Sample volume Elution volume Dilution volume (optional)

The following table gives an overview over purification protocols installed together with software version 3.0:

Purification Protocols pre-installed together with MagNA Pure LC Software 3.0


Name of Protocol in MagNA Pure LC Software 3.0 DNA I Blood_Cells High Performance DNA I Blood_Cells Fast DNA I High_Performance_ External_Lysis DNA II Tissue Application Sample Amount possible to process 20-200 l (102-106 cells) 20-200 l (102-106 cells) 20-200 l (102-106 cells) 110 mg tissue 110 mg tissue Input Volume Elution Volume 100 l Processing Time per 32 samples 92 min Used Reagent Kit

Isolation of high quality genomic DNA from blood or cells Fast Isolation of genomic DNA from blood or cells To process externally pre-lysed samples To isolate high quality genomic DNA from tissue samples Allows a variable protein digest outside of the MagNA Pure LC Instrument before starting the automated purification. This protocol must be used for formalin-fixed/paraffinembedded tissue. It is also useful for fresh/frozen tissue samples that are difficult to homogenize, like certain tumor samples.

200 l

MagNA Pure LC DNA Isolation Kit I MagNA Pure LC DNA Isolation Kit I MagNA Pure LC DNA Isolation Kit I MagNA Pure LC DNA Isolation Kit II MagNA Pure LC DNA Isolation Kit II

200 l

100 l

54 min

200 l

100 l

92 min

8090 l

200 l

118 min

DNA II Tissue External_Proteinase_K

100110 l

200 l

67 min

DNA III Bacteria DNA LV Blood_1000

To isolate high quality bacterial or fungal DNA Isolation of high quality genomic DNA from blood or blood cells

50100 l 1 ml

100240 l 1 ml

100 l 200 l

99 min 180 min

MagNA Pure LC DNA Isolation Kit III MagNA Pure LC DNA Isolation Kit - Large Volume

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Name of Protocol in MagNA Pure LC Software 3.0 DNA LV Blood_300_500 DNA LV Blood_20_200 DNA LV Cells

Application

Sample Amount possible to process 300500 l

Input Volume

Elution Volume 100200 l 100 l

Processing Time per 32 samples approx. 140 min approx. 140 min 140 min

Used Reagent Kit

Isolation of high quality genomic DNA from blood or blood cells Isolation of high quality genomic DNA from blood or blood cells Isolation of high quality genomic DNA from culture cells To isolate total RNA from blood and white blood cells To isolate high quality total RNA from culture cells To isolate high quality mRNA from blood samples To isolate high quality mRNA from externally pre-lysed blood samples To isolate high quality mRNA from culture and blood cells To isolate high quality mRNA from tissue To isolate high quality mRNA from up to 107 blood cells To isolate high quality total (viral) nucleic acid from blood, serum and plasma samples using a fixed elution volume To isolate high quality total (viral) nucleic acid from serum and plasma samples using a variable elution volume To isolate high quality total (viral) nucleic acid from externally pre-lysed serum and plasma samples using a variable elution volume To isolate high quality total (viral) nucleic acid from up to 1 ml of serum and plasma samples

300-500 l

MagNA Pure LC DNA Isolation Kit - Large Volume MagNA Pure LC DNA Isolation Kit - Large Volume MagNA Pure LC DNA Isolation Kit - Large Volume MagNA Pure LC RNA Isolation Kit I MagNA Pure LC RNA Isolation Kit II MagNA Pure LC mRNA Isolation Kit I MagNA Pure LC mRNA Isolation Kit I MagNA Pure LC mRNA Isolation Kit I MagNA Pure LC mRNA Isolation Kit II MagNA Pure LC mRNA HS Kit

20-200 l

200 l

up to 5 106 cells 20 200 l 102 - 106 cells 10 -100 l 10100 l

100 l

200 l

RNA I Blood_WBC_PBMC RNA II Culture Cells mRNA I Blood mRNA I Blood_External_Lysis mRNA I Cells mRNA II Tissue mRNA HS Standard

200 l 200 l 10100 l 310400 l

100 l 100 l 100 l 25 100 l 25100 l 50 100 l

83 min 87min 95 min 95 min

102 - 106 cells

300 l

95 min

110 mg 106-107 blood cells (WBCs, PBMCs) 50-200 l

880 l 1200 l

104 min

Total NA Serum_Plasma_Blood

200 l

100 l

90 min

MagNA Pure LC Total Nucleic Acid Isolation Kit

Total NA Variable_Elution_ Volume

50-200 l

200 l

50 100 l

90 min

MagNA Pure LC Total Nucleic Acid Isolation Kit

Total NA External_Lysis

50-200 l

350500 l

50 100 l

90 min

MagNA Pure LC Total Nucleic Acid Isolation Kit

Total NA LV Serum_Plasma

2001000 l

1000 l

50 100 l

99 min

MagNA Pure LC Total Nucleic Acid Isolation Kit Large Volume

Note: The major application for the "DNA I Blood_Cells Fast" protocol is Mutation Analysis where high yield and linear

scalability of yield over the whole sample volume range is not required. The "DNA I Blood_Cells High Performance" protocol is recommended when you are looking for high yield, and/or for linear scalability over the whole sample volume range, e.g., if you are performing quantitative PCR analysis.

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To select the appropriate Purification Protocol, do the following:


On the Sample Ordering Screen find the Protocol

selection field:

Move the mouse pointer to the name of the appro-

Note: If you access the Sample Ordering Screen the

first time from the Main Menu Screen, the Sample Protocol field displays the name 'None', which means that no protocol is selected. A protocol selection is saved, even if you leave the Sample Ordering Screen or the software itself. The next time you access the Sample Ordering Screen again, the name of the last selected protocol is displayed. If you want to start from an empty Sample Ordering Screen use the function "New Sample Ordering".
Click on the downward-arrow button, which is loca-

priate reagent kit, you want to use for the purification run ( e.g., Total NA if you use the Total Nucleic Acid isolation Kit). When you select the kit name a second drop-down menu appears offering the purification protocols available for that reagent kit (in case of the Total Nucleic Acid Isolation Kit these are the Serum_ Plasma_Blood, the External_Lysis, and the Variable_Elution_Volume protocol).

ted right from the Protocol name field. A drop-down menu appears:

Select the purification protocol that you want to perform by clicking on its name. The protocol name will appear in the Sample Protocol name field:

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To enter volume information, do the following:


Note: When selecting a purification protocol recommended default values for sample and elution volume are automatically

entered into the respective entry fields by the software. If you want to change these entries, follow the steps below.
Begin by entering the value for the sample volume that For every purification protocol a minimum and maxi-

is pipetted into the wells of the Sample Cartridge.


Note: If you select a purification protocol that is

based on an external pre-treatment of the sample (e.g., Total_NA External_Lysis) the entry field is designated Lysed Sample Volume. In this case you should not enter the volume of the primary sample but the volume of the complete lysate.

mum sample, elution, and dilution volume is defined. It is not possible to start a batch run if an entered volume exceeds the allowed range. The allowed volume range for a volume entry field is displayed if you position the mouse pointer over this field:

Enter the volume of Elution Buffer in which the

nucleic acid is eluted from the magnetic particles within the Elution Cartridge:

If you enter a volume which is out of the allowed range and you click the Start Batch button, a warning message will appear displaying the allowed volume range:

Optionally you may define a Dilution Volume:

For dilution of eluted nucleic acid samples, an additional amount of Elution Buffer is prepipetted into the wells of the Storage Cartridge. The maximum allowed Dilution Volume is 900 l. If you define a dilution volume an additional Reagent Tub will be needed for uptake of the Elution Buffer used for eluate dilution. This additional Reagent Tub is always a medium tub M30 and is placed in position 7 of the Reagent Tub Rack, when performing a DNA isolation run, or in position 8, when performing an RNA or Total Nucleic Acid isolation run. For the isolation of mRNA dilution of eluates is not possible.

Please refer to the table at the beginning of this section, which gives an overview over the purification protocols, for correct volume ranges.

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3.3

Specifying Sample Data


Important:

The names and positions of all samples filled into the Sample Cartridge have to be specified before starting a purification run. This can be done by entering new sample names or by loading a previously saved Sample Order File.
To specify new sample names, do the following:
On the Sample Ordering Screen click well A1 of the

In addition to sample data, the SOD-file contains protocol information, which will overwrite any protocol information previously specified.

Sample Cartridge Graphic. The selected well changes to yellow:


Note: The rows in the

Repeat steps - until all sample positions are recorded.


Alternatively, samples can be entered directly into the

Sample Cartridge are labeled 1-4 (vertically); in each row, the wells are labeled A-H from right to left (horizontally). Always start entering sample information with well A1.

Sample Order Table: Click into the Sample Name field of the sample position you want to add and type the sample name. Press <Return>/<Enter> and the sample will be added to the Sample order Table and the Sample Cartridge Graphic.
Important: Beginning with position A1 samples have

In the Sample Ordering Table the row for the respective sample position is added. The entry field 'Sample Name' is automatically activated:

to be entered in a continous sequence without any interruption. Otherwise an error message appears when clicking the Stage Setup button and it is not possible to start the purification run:

Into the Sample Name field enter a sample name by

either typing it using the keyboard, or using alternatively a barcode reader to scan the barcode on the sample.

Note: See Chapter A, section 3.2.3 for details on Bar-

code Readers available from Roche Applied Science.


Note: You may also use the "Ctrl + C" ("copy") keys

and the "Ctrl + V" ("paste") keys to add sample names to the appropriate fields.
Each sample in the Sample Cartridge must be named

individually, starting with the top row and naming the samples right to left. Once you have entered a name for the sample in position A1, enter a name for position B1, then C1, D1, ..., H1. Then, start entering data for positions A2, B2, ..., H2, and so forth.

Confirm by pressing OK and right align the samples within the Sample Cartridge.

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To load an existing sample order, do the following:


From the Sample Ordering Screen File menu choose If you choose the Load Sample Order option and the

the function 'Load Sample Order':


The following file selection window will appear:

Sample Order information is already entered the following warning message will appear:

If you want to keep the existing sample data click


Cancel.

If you want to load a Sample Order file confirm by clicking OK. Select the SOD-file you want to load by clicking on its file symbol, and confirm your selection by clicking the Open button or pressing <Enter>. The sample information from the Sample Order file will be loaded and displayed on the screen.

The directory Sample Order within the main directory of the MagNA Pure LC software will be opened automatically and previously saved Sample Order files (*.SOD) will be displayed.

To edit the Sample Order information, do the following:


If you want to change a sample name, left-click into

the Sample Name field within the Sample Order Table. A blinking cursor will appear. You can now use the usual keyboard functions to edit the name. If you want to mark a complete word in the sample name field double-click on it. The selected word will be higlighted black.
If you want to copy a sample name to paste it into one

If you use the function Cut, the sample name is copied and deleted from the selected sample name field at the same time.
To insert the copied sample name into another sample

name field, right-click into that field and from the appearing pop-up menu choose Paste.

or several other sample name fields, select the sample name as described under Step 1. Then right-click on the selected sample name. A pop-up menu appears offering several editing functions:

Click on Copy to copy the sample name.

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If you entered too many samples you can easily remo-

ve a sample row and the corresponding well in the Sample Cartridge Graphic by right-clicking on the number field. The editing pop-up menu appears. Choose 'Cut' to remove the sample from the Sample Name Table and the Sample Cartridge Graphic.

All editing operations can easily be applied to multiple sample lines at a time. Select a range of multiple lines either by dragging the mouse over them or by selecting the first one and, while holding the shift key, clicking on the last one.
If you want to check the position of a sample in the

Sample Cartridge left-click on the number field in the Sample Order Table. The corresponding well in the Sample Cartridge Graphic is displayed in green:
Note:

You may use the edit menu also to copy a complete sample and to add it to a different position in the Sample Cartridge.

To save a Sample Order, do the following:

From the Sample Ordering Screen File Menu choose

the option 'Save Sample Order' :


The following file selection window will appear:

The directory Sample Order within the main directory of the MagNA Pure LC software will be opened automatically.
Into the field 'File name' type the desired name for

the Sample Order file. The file extension .sod will be added automatically. Confirm by clicking the Save button or by pressing <Enter>.

If you want to print the Sample Order information, do the following:

From the Sample Ordering Screen File Menu choose

the option 'Print Sample Order':


The sample order information will be printed

automatically on the selected and installed default printer.

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4.

Preparation for Operation


This section describes how to use the Start Information Screen and how to place disposable plastics and reagents needed for the isolation run on the Reagent/Sample Stage.

After the user has selected the appropriate purification protocol and has entered all necessary volume and sample information the instrument is ready to be prepared for the batch run by placing needed disposable plastics, reagents, and the samples on the Reagent/Sample Stage.

4.1

The Start Information Screen

The software uses the sample information defined on the Sample Ordering Screen to calculate which disposable plastics and volumes of isolation reagents are needed. This information is displayed in form of the Stage Layout Graphic on the Start Information Screen. The Stage Layout Graphic is a schematic representation of the Reagent/Sample Stage. Every position of a required disposable plastic is represented by a text box. For Reagent Tubs and Reaction Tips type and detailed position of the disposable is indicated on the left side of the text box

(e.g., R5_M20: Reagent Tub Medium M20 in Rack Position 5; L1: Position 1 in the Large Reaction Tip Rack). In addition, for Reagent Tubs type and volume of reagent or buffer, that has to be filled into the tub, is displayed within the text box (e.g., Proteinase K, 1100 l). For the Sample Cartridge the specified amount of Sample Volume is displayed. Use the Stage Layout Graphic as a guide to place the correct number of disposable plastics and the correct volume of reagents on the Reagent/Sample Stage.

To view the Start Information Screen, do the following:

On the Sample Ordering Screen click the function

button Stage Setup :

Note: It is not possible to proceed to the Start Infor-

mation Screen unless all necessary protocol and sample information has been entered correctly on the Sample Ordering Screen. In this case, after clicking the Start Batch button, a message window will appear, which informs about missing or incorrect entries.
The Start Information Screen will appear displaying

the Stage Layout Graphic. Use the Stage Layout Graphic as a guide to place the correct number and kind of disposable plastics, and to fill the right volume of isolation reagents:

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Menu Bar of the Start Information Screen File Menu


Print Cartridge Barcode Generates and prints a barcode label (if an optional barcode printer is installed) which can be used to track and identify the Sample and Storage Cartridge of the current experiment (see also Sample Ordering). Close Closes the Start Information Screen and returns to the Sample Ordering Screen.

Actions Menu
Change Dropcatcher Allows replacement of the disposable plastic (Tub Lid Seal) on the Liquid Drop Catcher. After clicking this button, the robotic arm moves to a position above Processing Cartridge I and the Liquid Drop Catcher located on top of the magnetic plate moves forward. The front door opens and you can now exchange the Liquid Drop Catcher disposable plastic. See Chapter B, section 2.4 for details about changing the disposable plastic of the Liquid Drop Catcher. Home By clicking the Home button the robotic arm is moved back to its Home position after exchanging the disposable plastic (Tub Lid Seal) on the Liquid Drop Catcher. Cover Lock Locks the instruments cover. The batch run can only be started if the cover is locked. Use this function to lock the cover after exchanging the Liquid Drop Catcher disposable.

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Graphical Elements of the Start Information Screen


Stage Layout Graphic Shows the required type and number of disposable plastics and volumes of reagents needed. After placing all required reagents and disposable plastics into their appropriate positions on the Reagent/Sample Stage, confirm the correct placement by clicking on the respective text boxes. The text boxes 'Samples', 'Elution', and 'Storage' represent the positions for Sample, Elution, and Storage Cartridge, respectively. For easier identification and to avoid any mix up of reagents the Reagent Tub text boxes are underlayed with the color of the corresponding reagent bottle lid. The same colors are found on the Positioning Frames available for placement on the Reagent Tub rack (see Chapter B, section 4.2.5 for details). To avoid mix up of the Reagent Tubs M20 and M30, the label for M30 tubs is displayed in bold, blue letters. Note: The text box Liquid Waste Bottle / Liquid Waste Funnel is displayed only if the option Liquid Waste Discard has been activated on the Sample Ordering Screen. The text box Cool Block 2 is displayed only if a Post Elution protocol has been selected on the Sample Ordering Screen. The purification run can only be started after all positions have been confirmed.

Instrument Status Indicators

Cover Status / Cover Lock Status: Display the open and lock status of the instrument cover door. Both indicators have to be on green to start the purification run. Lock Bar Status: Displays the status of the Dispo Lockbar (red = open, green = closed). The batch run can only be started if the Dispo Lockbar is closed. See Chapter B, section 2.3.2 for details on handling of the Dispo Lockbar. Heat Unit Status / Cool Unit 1/2 Status: Display the thermal status of the respective units (red = fail: The target temperature has not been reached yet; green = pass: The target temperature has been reached). All indicators have to be on green to start the batch run.

Protocol Indicator

Displays the name of the active purification protocol.

OK Button

Click on this button to start the batch run. The button is visible only after placement of all needed disposable plastics and reagents has been confirmed. Note: The button is active also when the Heat and Cooling Units have not yet reached their final target temperature. You are able to start the run immediately but the actual process will not start before the required Heating and Cooling Unit temperatures are reached.

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Under unfavourable circumstances (e.g., instrument has been turned on only shortly before starting the run, or high ambient temperature) it might take maximally up to 25 min until the final target temperature is reached, beginning with the selection of the respective protocol. Normally, when you select your protocol first, until you are ready to place the magnetic beads on the MPLC, there is only a short period of time left, until the machine will start and this will be perfectly ok. However, in case you have to wait for the full 25 minutes, the magnetic particles within the Reagent Tub during this time period, may sediment, possibly leading to clumping of beads during the isolation process. Furthermore, alcohol may evaporate from alcohol-containing buffers. Altogether this may lead to a decrease of nucleic acid yield. Thus, it is recommended to select the respective protocol before setting up the reagents and samples on the instrument and to always place the magnetic beads on the MPLC last.

4.2

Placing the Disposable Plastics on the Reagent/Sample Stage


Place Storage Cartridge into Cooling Unit 1 Place Elution Cartridge into Heating Unit Place Processing Cartridge(s) Place Tip Stand(s) (if required) Place Large the instrument Fill reagents into Reagent Tubs (outside the instrument) Close Reagent Tubs with Reagent Tub Lids (outside the instrument) Place filled Reagent Tubs (within rack) Fill samples into Sample Cartridge (outside the instrument) and Small Reaction Tips Place Reagent Tubs into Reagent Tub Rack outside

Use the information of the Start Information Screen to place the disposable plastics and reagents necessary for the following batch run on the Reagent/Sample Stage. It is recommended to fill the Reagent/Sample Stage in the following order:

Place Sample Cartridge


Note: If you want to perform a Post Elution run directly fol-

lowing the purification run, you may already place MagNA Pure LC Cooling Blocks into Cooling Unit 2 before starting the purification run. Please follow the instructions given in Chapter B, section 2.3.5. The following sections describe the correct placement of disposable plastics and reagents in detail.
Note: Gloves should be worn during all handling of dispos-

ables and reagents, all manipulations steps inside the instrument, and all manually performed pipetting steps. Always use sterile, nuclease-free pipettes.

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4.2.1 Placing Elution and Storage Cartridge (Stage position 10/11)

The same disposable plastic is used as Sample, Elution, and Storage Cartridge (see Chapter A, section 4.3 for details). For use as Elution Cartridge it is placed in the Heating Unit (stage position 10), for use as Storage Cartridge of the eluted nucleic acids it is placed in Cooling Unit 1 (stage position 11). Place the disposable as indicated in the figure below. Under the back rim of each cartridge, there are spac-

er fins (to allow easy removal of the cartridge). Two of these fins (under rim on the left side) are longer than the other fins. Insert these longer fins into the slots on the left side of the blocks. If you reverse orientation of the cartridge (so longer fins are on the right), the cartridge cannot be inserted correctly.

Top View of Elution/Storage Cartridge: Correct orientation


Slots to ensure correct orientation

Corner cut for positioning

Incorrect

Caution:

To avoid collisions between the robotic arm and the sample cartridges, always check correct placement of cartridges.
4.2.2 Placing the Processing Cartridges (Stage position 9)

There are four positions on the Reagent/Sample Stage for Processing Cartridges. Just insert each cartridge into one of these positions. Each Processing Cartridge is symmetrical and does not have to be placed on the Reagent/Sample Stage in a particular orientation. Each cartridge is used for a purification process of 8 samples. Every process has a fixed Processing Cartridge position: The first eight samples (Process 1) are processed in the cartridge placed in the front left position, the second eight (Process 2) in the cartridge placed in the front right position, the next eight (Process 3) in the rear left, and the last eight (Process 4) in the rear right. Be sure to place the Processing Cartridges in the correct order when running fewer than four processes.

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4.2.3 Placing the Tip Stands (Stage position 8)

There are five slots on the Reagent/Sample Stage for Tip Stands (which are used to store used Reaction Tips temporarily). Four Tip Stands are located behind the Processing Cartridges: these store protocol execution tips. One Tip Stand is located alone at the rear behind the Tip Stand of Processing Cartridge 4. This one stores tips during reagent dispensing, if sample numbers not divisable by eight are processed. The slots for the Tip Stands are not ordinary rectangular holes, but are butterfly shaped. Inside each slot, there are two metal springs (at the left back and right front of slot). These springs keep the Tip Stand parallel to the X-axis.

To place the Tip Stands: 1. Insert a Tip Stand into a butterfly-shaped slot. 2. Keep pushing down on the Tip Stand while turning the stand clockwise, until the stand touches the aluminum surface of the Reagent/Sample Stage. Result: The Tip Stand spontaneously turns parallel to the X-axis. Then, the right upper and left lower corners of the stand insert into hooks at the corner of the slot. These hooks prevent the Tip Stand from lifting with the tips when the Nozzle Head picks up the parked tips.
Note: To remove the Tip Stand, turn the stand counter-

clockwise and pull it up.

Butterfly-shaped slot for Tip Stand

Hooks to secure Tip Stand

Insert a Tip Stand

Turn clockwise and push down

Stand spontaneously inserts into hooks

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4.2.4 Placing the Reaction Tips (Stage position 3/4)


Important: Before you can place Reaction Tip Trays, Re-

agent Tubs, and the Sample Cartridge on the Reagent/ Sample Stage, you must open the Dispo Lockbar (by flipping the handle on the right side of the Lockbar up) and lift it out of the way (see Chapter B, section 2.3.2). After placing the Reagent Tubs in the Tub Rack, close the Dispo Lockbar and lock it (by pushing the handle on the right side of the Lockbar down). Start of the purification run is not possible if the Dispo Lockbar is open. The MagNA Pure LC Instrument uses two different sizes of Reaction Tips (large and small). Both sizes are supplied either in pre-packed Tip Trays (filled with 32 tips) or bulk packed in Refill Packages (for refilling Tip Trays). The Tip Tray for large tips is blue and the one for small tips is yellow. Place the number of Tip Trays indicated on the Start Information Screen in their appropriate Tip Tray Rack: Large

(blue) reaction tips are put into the blue rack in the rear position (stage position 3), small (yellow) tips are put into the yellow rack in the front position (stage position 4). The positions for Tip Trays containing large tips are designated (back to front) L1, L2, and L3 on the Start Information Screen, those for small tips S1, S2, and S3.
Notes: The intended use of the Reaction Tips in Refill Pack-

ages is to refill the Tip Trays only if a small number of Reaction Tips was used during a purification run. It is not recommended to fill completely empty Tip Trays with tips from Refill Packages, because this poses the danger of contamination. The instrument uses Reaction Tips from L1 first, then from L2, and finally from L3. When refilling the Tip Trays from a Refill Package, always refill the back tray (L1) first, then L2, and finally L3.

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4.2.5 Placing the Reagent Tubs (Stage position 2)

It is recommended to fill the Reagent Tubs with reagent and to place them in the Reagent Tub Rack outside the instrument, to avoid any contamination of the Reagent/Sample Stage.

Place the decontaminated Reagent Tub Rack on a clean bench. Insert each reagent tub as indicated by the Start Information Screen into the rack. There are eight positions for reagent tubs in the rack: Two for Large Reagent Tubs (R1, R2) and six for Medium/Small Reagent Tubs (R3-R8).
back

The type of Reagent Tub is also indicated by the Start Information Screen: L = Large Reagent Tub M20 = Medium reagent Tub M20 M30 = Medium Reagent Tub M30 S = Small Reagent Tub

R1 large R2 R3 R4 R5 R6 R7 R8

medium/ small

front

For correct positioning insert the fins of the Reagent Tub (located under the left and right rim) into the slots of the rack.

Note: It is recommended that you fill only the exact

Fill the reagent indicated by the Start Information Screen inside the text box of the appropriate reagent tub. Note: To avoid any mix up of reagents the color code of the corresponding reagent bottle is additonally displayed on the Start Information Screen. To avoid mix up of the Reagent Tub M20 and M30, the label for M30 tubs is displayed in bold, blue letters. Important: Use only sterile, nuclease-free pipettes

volume of reagents indicated by the Start Information Screen, sufficient for the following run. On the other hand, you could also fill excess reagent sufficient for more than one purification run. Finally, you could store the reagents being left over after completion of the first run, in the Reagent Tub. If you want to store excess reagents in the Reagent Tubs, always close them with a Reagent Tub Lid Seal. The Tub Lid Seal is suitable for Large, Medium, and Small Reagent Tubs, and fits into the recess of the Reagent Tub Lid.

continued on next page

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The following precautions have to be taken into account:

The Tub Lid Seals do not close the Reagent Tubs airtight. Because of this, it is recommended to store reagents being left over after a purification run only for hours or overnight, but not for days. Evaporation of liquid may lead to change of buffer composition thereby causing incorrect purification results. Reagents should be stored at room temperature to avoid cristallization, except Proteinase K solution, which must be stored at 4C. Before starting a purifcation run make sure that all reagents are equilibrated to room temperature. If precipitates have formed during storage, warm the reagent before use at 20 - 37C with gentle shaking until precipitates have dissolved. Magnetic particles should never be stored in the Reagent Tub, because they tend to sediment and clump. Fill the amount required for only one purification run. If placing a stored reagent/buffer contained in a Reagent Tub back onto the Reagent/Sample Stage for the next purification run, it is important to remove

the Lid Seal. Otherwise the reaction tips will hit the seal during pipetting which will cause the instrument to stall the purifcation run. After filling the reagent always close the Reagent Tub using the appropriate Reagent Tub Lid, to avoid evaporation of liquid during operation. Place the Reagent Tub Rack into Reagent/Sample Stage position 2 as described in detail in Chapter B, section 2.3.3.
Important:

Always fill magnetic particles (Magnetic Glass Particles, Streptavidin Magnetic Particles) as the last reagent shortly before the start of the isolation run, because they tend to sediment fast and are stored in isopropanol, which easily evaporates. Before pipetting Magnetic Glass Particles into the Reagent Tub, vortex them vigorously to ensure a completely homogeneous suspension. Clumping of magnetic particles may lead to malfunction and decreased yield of isolated nucleic acids. Do not vortex Streptavidin Magnetic Particles used in the mRNA isolation kits. Instead, suspend them homogeneously by using a roller incubator.

Using Positioning Frames to ease Placement of Reagent Tubs and Reagents

For every MagNA Pure LC isolation kit a Positioning Frame for the Reagent Tub Rack is available. These Positioning Frames fit on the rim of the Reagent Tub Rack. The Positioning Frames show the correct order of reagents in the

Reagent Tub Rack for the respective reagent kit. The name of the reagent is printed on the left border of the frame, while the right border shows the color code of the reagent vessel from the reagent kit.

Positioning Frames may be ordered separately. See the following table for ordering details:
Positioning Frame for MagNA Pure LC isolation kit DNA Isolation Kit I DNA Isolation Kit II DNA Isolation Kit III DNA Isolation Kit - Large Volume RNA Isolation Kit I RNA Isolation Kit II mRNA Isolation Kit I mRNA Isolation Kit II mRNA HS Kit Total Nucleic Acid Isolation Kit Total Nucleic Acid Isolation Kit - Large Volume Ordering Number 2 256 096 3 142 663 3 262 758 3 328 465 2 256 100 2 256 118 2 256 126 3 142 655 3 274 093 2 256 134 3 262 863

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As an example the Positioning Frame for the MagNA Pure LC DNA Isolation Kit I is shown:
name of the reagent kit

name of the reagent for this tub position

lid color of the reagent vessel from the kit

ordering number

4.2.6 Stage position 1: Sample Cartridge

Fill the samples to be isolated into wells of the Sample Cartridge. Bear in mind the correct order of pipetting:
direction of sample pipetting

Note: For some sample materials a specific pre-treatment

is necessary before they can be placed in the Sample Cartridge for automatic purification. Please refer to the instructions in the pack insert for the respective MagNA Pure LC isolation kit used.
Important: Do not pipette samples directly into the

first well

Sample Cartridge already inside the instrument. This poses the danger of contamination.

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4.2.7 Placing the Waste Bag

When the instrument has finished using a set of Reaction Tips, it automatically discards them through the Waste Disposal Slide into the plastic Waste Bag, that fits over the slide (see Chapter A, section, 4.3 and Chapter B, section 2.3.4 for details). To install the Waste Bag on the Tip Waste Disposal Slide:
. Remove the Tip Waste Disposal Slide and place a

. Insert the Tip Waste Disposal Slide into its notches

on the front of the instrument.


. Use the magnet to hold the front of the bag on the

Tip Waste Disposal Slide.


Caution:

plastic bag over the mouth of the slide.


. Ensure the bag covers the hook on the back of the

Do not insert the slide upside down! Make sure the hook on the back of the slide engages the correct notches and that the bag is trapped firmly between hook and notches. See Chapter B, section 2.3.4 for a detailed description of Waste Tip Slide positioning.

slide.

Hook to secure bag

Magnet for holding bag

Hook inserted into notch, trapping a bag.

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4.2.8 Placing the Liquid Waste Bottle

If you select the optional "Liquid Waste Discard" function in the Sample Ordering Screen, the instrument automatically discards the liquid left in the wells of the Processing Cartridges after completion of the purification run. To store the liquid waste, you must place a disposable Liquid Waste Bottle on the Waste Bottle Tray and insert the Liquid Waste Funnel into the opening of the bottle.

Important: The instrument does not automatically check

the liquid level inside the Liquid Waste Bottle. Always ensure that there is enough volume for uptake of liquid waste. Since the bottle can keep the liquid waste for one full batch preparation (approx. 260 ml), replace the bottle with an empty one after each full run.

There is a removable Waste Bottle Tray under the bottle to catch any overflow. Overflowing liquid from the bottle is temporarily stored in the tray. However, if the overflow is too great, liquid will flow onto the lab bench.
Note: As shown in the figure beside, you may also place the

Waste Bottle on the Waste Bottle Tray outside the instrument, and then slide both the tray and the bottle into the Waste Bottle compartment of the instrument.

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4.3

Confirming the correct Setup of the Reagent/Sample Stage

Before you are able to start the purification run, you must confirm the correct placement of all disposable plastics and reagents on the Reagent/Sample Stage by mouse-clicking on the text boxes of the Start Information Screen representing the needed items. After clicking on a text box its display will change to a schematic figure of the respective disposable plastic or

accessory. For the Reagent Tubs the color code of the corresponding reagent bottle from the kit is shown. After clicking all text boxes the Start Information Screen may look as follows (display changes with Purification Protocol used and number of samples specified).

Important:

Make sure that all disposable plastics are correctly placed and no other obstacles are on the Reagent/ Sample Stage.
Do not omit this step!

For safety reasons, the robotic arm performs a slow surface scan at the beginning of a purification run to identify any obstacles (e.g., improperly seated disposables or cooling blocks, items accidentally left on the Reagent/Sample Stage). The software does not check whether the Reagent/Sample Stage is equipped correctly with disposable plastics and reagents. It relies on your visual inspection of all needed items. Ensure the correct item is present before clicking the corresponding text box on the Stage Layout Graphic.

After having confirmed all text boxes and having closed and locked the Dispo Lockbar as well as the instrument cover (the respective status indicators display 'green') the 'OK' button is displayed. The purification run can be started now.
Note: The 'OK' button is displayed even if the Heat and

Cooling Units have not yet reached their target temperature (the respective status indicators display 'red'). In that case, you can initiate the purification run but the actual automated process will not start before the final temperature is reached. As this is controlled autonomously by the software you may leave the instrument after clicking the 'OK' button.

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5.

Monitoring the Purification Run on the Batch Status Screen


The Batch Status Screen displays the total timeline of the purification run and the status of the active isolation processes in a graphical way:

After clicking the "OK" button on the Start Information Screen the purification run starts and the Batch Status Screen is shown.

During the Prologue phase the surface scan of the Reagent/ Sample Stage is executed and reagents are transferred from the Reagent Tubs into the wells of the Processing Cartridges. For every eight samples an individual purification process is performed corresponding to one Processing Cartridge. Working periods of the process are shown as blue boxes, incubating periods as white boxes. Note: Some purification protocols perform Staggered Processing to safe time (e.g., DNA I High_Performance protocol). With Staggered Processing two purification processes are run in parellel: One Processing Cartridge is processed, while the other one is incubating. Staggered Processing is only possible for two processes at the same time. In case 32 samples are processed, the first 16 samples are processed in two staggered processes. After completion of Process 2, the following 16 samples are processed in two further staggered processes. During the Epilogue phase the (optional) Liquid Waste Discard is performed. The name of the purification protocol currently under process is displayed.

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The red line shows the current position in the purification process.

The time axis shows the current time and expected completion time of the purification run. In the status line the current number of samples flagged as "Fail" (e.g., due to detection of a clot) is displayed.

Manually Stopping a Batch Run


It is possible to stop a purification run manually. To avoid that a run is aborted inadvertantly, the Stop Batch function has to be activated first by choosing the Activate Stop Batch option from the Actions menu. Only then the function 'Stop Batch' will be accessible. The run is only stopped when you additionaly choose the option 'Stop Batch' from the Actions menu. Note: Reaction Tips are discarded automatically after a run was stopped manually. Important: The batch run will be stopped immediately without any safety query!

It is not possible to resume the run after stopping it manually!

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6.

Evaluation of the Purification Outcome on the Result Screen


Note: If you have selected a Post Elution protocol on the

After completion of the purification run the Result Screen appears automatically. The Result Screen shows the sample and protocol information previously entered on the Sample Ordering Screen. In addition, the result status ("Pass" or "Fail") for every sample is shown.The information from the Result Screen may be printed or saved as file. If an error occurred during the purifcation run, information from the Error Log Files can be viewed or printed, to obtain helpful information for troubleshooting.

Sample Ordering Screen, this protocol is processed directly after completion of the purification run and the Result Screen is not shown in between. After completion of the Post Elution run, first the Result Screen is shown, only then the Post Elution Result Screen.

All operations that can be performed from the Result Screen are initiated from the screens menu bar:
Menu Bar of the Result Screen File Menu
Load Result Screen Opens a previously saved Result Screen file. Save Result Screen Saves the information from the Result Screen as file (*.ird). Print Result Screen Prints information displayed on the Result Screen: Print Sample Protocol Prints a protocol of the working steps performed during execution of the last purification protocol: Printer Setup Lets you choose appropriate printer driver and printer settings. Close Closes the Result Screen and returns to the Sample Ordering Screen.

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Actions Menu
Open Post Elution Opens the Post Elution Steps Screen for programming and starting a Post Elution protocol. Sample information is transferred automatically from the Result Screen to the Post ElutionSteps Screen.

Help Menu
Note: The Help menu items are found with identical function on every screen, and thus are only explained once at this point. Help Opens the Operators Manual pdf file (to view the file Adobe Acrobat Reader 4.0 or higher has to be installed). View Error Log / Print Error Log Displays or prints a list of previous errors and warnings. The error log file stores sequentiallly all error messages which might appear during operation of the instrument. The last 50 errors are registered together with a summary of instrument-specific data and the date at which each error occurred. Information from the error log file may be helpful for the service engineer or technical support to track down the cause for a certain instrument malfunction. See Chapter B, section 9.6 for details about saving the error log files onto a floppy disk. About Opens a window displaying software version and system information.

Graphical Elements of the Result Screen Result Table


In the Result Table all sample information (name, comment, position) entered previously on the Sample Ordering Screen is displayed. In addition, the Result column of the table indicates the status of each sample after completion of the purification run:

Pass/Green: The sample was purified correctly Fail/Red: During purification of this sample an error occurred

For samples flagged as "Fail" the respective error code is displayed (e.g., E54, if the sample was clotted). Please refer to to Chapter C, section 1 for a detailed description of error codes. Note: The indication of a failure for a specific sample does not mean that this sample cannot be used further or even has to be discarded. The indicator "Fail" only points towards any problem that occurred during the purification run. The actual state of the nucleic acids prepared from such a sample can only be detected by following experimental analysis. On the other hand, experimental data from "failed" samples should always be rated critically.

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7.

Creating and Starting a Post Elution Protocol


Further dilute isolated nucleic acid samples Create dilutions series for e.g., concentration

In addition to its usage as a workstation for automated nucleic acid preparation, the MagNA Pure LC system can be used as a programmable pipetting robot, e.g., for setup of downstream PCR/RT-PCR reactions, using the previously isolated nucleic acids as template. Because reaction setup is performed after completion of the nucleic acid purification run, i.e. after elution of the isolated nucleic acids, this process is called "Post Elution". But it is not necessary to perform a Post Elution run in direct combination with a purification run: A Post Elution run can also be performed as a single process using previously isolated and stored nucleic acid samples. All pipetting steps performed by the instrument are programmed by the user and stored as Post Elution Protocol file. Use the Post Elution function of the MagNA Pure LC system to execute the following tasks: Add template nucleic acid to PCR master mixes in PCR reaction vessels (e.g., LightCycler capillaries or 96-well PCR plates)

standards
Combine PCR reagents to PCR master mixes Archive eluted samples into Reaction Tubes for

further storage The following sections describe how the Post Elution software module is started how a new Post Elution Protocol is programmed how a Post Elution Protocol is edited how a pre-programmed Post Elution Protocol is saved and loaded how a Post Elution run is started how the result of a Post Elution run is viewed how information from the Post Elution Result Screen is transferred via a SAM-file to the 'Edit Samples' Screen of the LightCycler software.

7.1

Opening the Post Elution Steps Screen

Programming of a new Post Elution Protocol, loading of existing Post Elution Protocols, and start of a Post Elution run is done on the Post Elution Steps Screen. The Post Elution Steps Screen is accessible from three points within the MagNA Pure LC software: On the Standard User Main Menu Screen click the 'Post Elution Handling' Button. On the Sample Ordering Screen or the Result Screen choose the function 'Open PE Handling' from the Actions menu. Regarding handling of sample data the following possibilities exist: No sample information is specified in the Sample Ordering Screen the user enters necessary sample information directly on the Post Elution Steps Screen or loads a previously saved Post Elution file and uses the sample information saved within.

Sample information is specified on the Sample Ordering Screen or the Result Screen sample information is transferred to the Post Elution Steps Screen

Important: For starting a Post Elution Protocol do not sel-

ect a previously stored Post Elution Protocol from the respective pull-down menu in the Sample Ordering Screen. This function is used only for automatic start of a Post Elution run in direct combination with a purification run. You must be aware, that in this case there will be no pausing between the purification run and the Post Elution run. Therefore all disposable plastics and reagents needed for Post Elution should be placed on the Reagent/Sample Stage in advance.

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7.2

The Post Elution Steps Screen

No Post Elution Protocol loaded

Post Elution Protocol loaded

Menu Bar of the Post Elution Steps Screen Note: Some functions are only accessible if a Post Elution protocol is loaded.
File Menu New Post Elution To program a new Post Elution protocol. Existing screen data will be lost after confirmation. Load Post Elution To load a previously saved Post Elution Protocol file (*.pep).

Note: MagNA Pure LC software version 3.0 stores Post Elution


protocol information within a single file (*.pep). Software versions prior to v3.0 store Post Elution protocol information in two separate files: *.pep and *.pes. While the pep-file contains information on used Cooling Blocks and positions, the pes-file contains information on the actual Post Elution pipetting steps. Therefore, if you want to distribute a Post Elution protocol created with a software prior to v3.0, always copy both file types. If you load a Post Elution protocol file created with software version 2.1 into software version 3.0 it will automatically be converted into the new format. To keep the new format you have to save the protocol file. Save Post Elution To save a new or modified Post Elution protocol. Delete Post Elution Deletes the Post Elution protocol file that is currently loaded from the harddisk. Print Post Elution Prints the list of Post Elution pipetting steps displayed in the Protocol Description Table. Printer Setup Lets you choose an appropriate printer driver and printer settings.

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Print Cool Block Barcode Prints a barcode label (if an optional barcode printer is installed) which can be used to track and identify the samples pipetted by Post Elution. If performed in combination with a prior purification run the Cool Block barcode will be identical to the BatchID of the purification run. This function is especially useful during A-Ring workflow: If you do not want to use the original A-Ring barcode(s) you can create your own barcode(s) using this function. If performed in combination with a prior purification run, the Cool Block Barcode will be identical to the BatchID, with the figure 1 added for the left A-Ring Cooling Block part, and the figure 2 added for the right A-Ring Cooling Block part. The barcode can subsequently be read-in for naming of a Post Elution Result file (*.per) (if an optional Barcode Reader is installed). View Result Loads a previously saved Post Elution Result file (*.per) Close Closes the Post Elution Steps Screen and returns to the Main Menu Screen or the Sample Ordering Screen depending from which screen it was opened.

Edit Menu Note: The Edit Menu is also shown when you right-click on the Post Elution Steps Table. Some of the functions are also activated by using the displayed key combinations. Cut Removes one or several selected protocol steps from the Protocol Description Table. The programming step data are stored in clipboard memory and may be pasted to a different protocol position. Copy Copies programming step data from one or several selected protocol steps to clipboard memory which then may be pasted to a different protocol position. Paste Inserts previously copied or cut protocol step data before a selected protocol step. Paste After Inserts previously copied or cut protocol step data after a selected protocol step. Insert a Blank Row Inserts a blank row into the Protocol Description Table before a selected programming step.

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Insert a Blank Row After Inserts a blank row into the Protocol Description Table after a selected programming step. Delete Row(s) Deletes one or several selected programming steps from the Protocol Description Table. Undo Use this function to revoke the last three programming or editing actions.

Actions Menu Define Name / Set Volume Opens an input window for defining type, name and initial volume of reaction vessel positions for selected cooling blocks. Start Post Elution Starts the Post Elution pipetting process.

Graphical Elements of the Post Elution Steps Screen


File Path If a Post Elution Protocol file is loaded its file path is shown.

Protocol Description Table In the Protocol Description Table the programmed AspirateDispense sets of the Post Elution Protocol are listed. During execution of the protocol these steps are processed from top to bottom. The column Description shows the name of the sample/ reagent container (e.g., Sample Cartridge or LC Carousel Cooling Block): left column corresponds to the Aspirate From position, right column corresponds to the Dispense To position. The column L/R indicates whether a cooling block is positioned in the left or right half of Cooling Unit 2 (only when using small Cooling Blocks). The column Position displays the position of the reaction vessel in the corresponding container (Sample Cartridge or Cooling Block). The column Volume shows the volume of sample/reagent that is pipetted from or to the corresponding vessel. Note: It is possible to change the column width by moving the mouse pointer over a column divider line in the column title area and dragging it to a new position. To indicate its change of functionality the mouse pointer changes from a cross to two vertical lines when it is positioned over a divider line.

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Each Aspirate-Dispense set consists of at least two rows: A status line displaying general information on mixing and tip discard status, and an parameter line displaying the parameters of the Aspirate-Dispense Set (Aspirate From position, Dispense To position, Dispensing Volume), or Mixing Step (Mixing Volume), respectively. Information on the 'Aspirate From' position is always shown in the left half of the table, while information on 'Dispense To' positions is displayed in the right half. Type and status of Aspirate-Dispense sets are indicated by the color of the status line: Common Aspirate-Dispense set, no error Volume error due to a too low initial volume or because of exceeding the maximum capacity of a reaction vessel mixing step pausing step

Heating Unit Graphical display of the Heating Unit. Note: In the current version of the MagNA Pure LC software positions from the Heating Unit cannot be included into a Post Elution Protocol.

Storage Cartridge Graphical display of the Storage Cartridge within Cooling Unit I. Samples processed during a previous purification run or otherwise specified are shown in yellow (shaded yellow if a volume is to be dispensed from this position during Post Elution.).

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MagNA Pure LC Cooling Block Graphical display of the selected MagNA Pure LC Cooling Block placed in Cooling Unit 2 (as an example the LC Sample Carousel Cooling Block is shown). Type and status of cooling block positions are indicated by color coding: Type and status of Cooling Block positions are indicated by color coding:

Empty position Position containing a reagent Position containing a sample Position which received a reagent and a sample volume Position which received volumes from two samples

The following labels can only be seen if the corresponding Aspirate-Dispense set is selected in the Protocol Description Table:

From this position a volume is aspirated Into this position a volume is dispensed Note: If for a position an Aspirate From volume is defined, the color code of this position is displayed shaded.

Programming Area The function buttons and input fields of the Programming Area are used for programming the Post Elution pipetting steps. Dispensing Volume: Enter the volume which should be dispensed from a reaction vessel (allowed range 5-100 l) Aspirating Volume: Cannot be modified in the current version of the software. Is automatically filled with the Dispensing Volume value Mixing Cycles: When programming a mixing step enter the number of mixing cycles (maximum number is 15) Discard Tips: Mark the checkbox if you want to discard the Reaction Tips after completion of the pipetting step. Note: When performing a multi-dispensing step and Discard Tips is activated Reaction Tips are discarded after every single pipetting step. If Discard Tips is not activated Reaction Tips are discarded automatically after the last pipetting step.

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Control and Information Bar In the Control and Information Bar the Start button for starting the Post Elution run, as well as the Pause / Stop button for stopping or pausing the Post Elution run is located (only accessible during an active Post Elution run). The number of required Large and Small Reaction Tips as well as the Estimated Total Time needed for completion of the Post Elution run is displayed. Note: Large Reaction Tips are currently used for mixing steps only. During the first run of a Post Elution protocol the actual total time needed for completion of the run is measured exactly. This Measured Total Time (MTT) may be saved together with the Post Elution protocol. If you load this protocol the next time, the MTT instead of ETT will be displayed in the Control and Information Bar. Status Line The Status Line displays information on a sample or reagent position (container, name of position, initial volume, added reagent). To view this information, place the mouse-pointer over a reaction vessel position.

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7.3.

Programming a new Post Elution Protocol

To program a new Post Elution protocol choose the option 'New Post Elution' from the File menu. Before you can program the actual pipetting steps of a Post Elution protocol you have to select the MagNA Pure LC

Cooling Block(s) that are placed in Cooling Unit 2. After Cooling Block selection, type, name, and initial volume of the reaction vessel positions in the Cooling Block have to be defined.

The following flow chart gives you an overview over the principle Post Elution programming workflow:

1) New Post Elution Choose New Post Elution from the File menu:

Choose between small MagNA Pure LC Cooling Blocks: LC Centrifuge Adapters Reagent Tubes PCR Plate A-Ring LC Sample Carousel 3) Define Name / Set Volume The Define Name / Set Volume window opens automatically 1. Specify Type of reaction vessel positions Reagent Sample/Standard Control 2) Select Cooling Block The Select Cooling Block window opens automatically

or large MagNA Pure LC Cooling Blocks

2. Specify Initial Volume of reaction vessel positions

4) Programming

a) Aspirate-Dispense Sets 1:n single dispensing 1:n multi-dispensing 1:1 multi-dispensing

b) Mixing

Select Aspirate From position Select Dispense To position Specify Dispensing Volume Confirm (Done)

Select Aspirate From position Choose Mix Specify Mixing Cycles Confirm (Done)

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7.3.1 Selecting MagNA Pure LC Cooling Blocks

When you choose the option 'New Post Elution' from

the File menu, the 'Select Cooling Block' window opens automatically. On this window, select the MagNA Pure LC Cooling Blocks that are placed into the large Cooling Unit 2. See Chapter A, section 3 for description of MagNA Pure LC Cooling Blocks and Chapter B, section 2.3.5 for details on how to place cooling blocks into Cooling Unit 2.

Use the 'Small Cooling Block' menu to select small MagNA Pure LC Cooling Blocks. Small cooling blocks you can

choose from are:


Name in Select Cooling-Block window MagNA Pure LC Cooling Block LC Centrifuge Adapters Reaction Tubes PCR-Plate, Type 1 PCR-Plate, Type 2 CoolingBlockARing(left) CoolingBlockARing(right) MagNA Pure LC Cooling Block, LC Centrifuge Adapters MagNA Pure LC Cooling Block, Reagent Tubes MagNA Pure LC Cooling Block, 96-well PCR Plate MagNA Pure LC Cooling Block, 96-well PCR Plate MagNA Pure LC Cooling Block, A-Ring Cat. No. 2 190 664 2 189 666 2 189 674 2 189 674

Note: The same cooling block is used for PCR-Plate, Type 1 and ,PCR-Plate, Type 2. The difference between both PCR-Plate types is depth of immersion of the Reaction Tips into the wells of the plate. See the following table for an overview over selected PCR tubes and PCR plates compatible to the MagNA Pure LC Cooling Block, 96-well PCR plate: Company Roche Diagnostics GmbH Applied Biosystems Applied Biosystems Eppendorf Eppendorf Eppendorf Sarstedt Item Thin-walled PCR Tubes (200 l) Micro Amp. Optical 96-well plate*) Micro Amp. Optical tubes*) 0.2 ml, PCR-plate, 96-well 0.2 ml, PCR-tube 0.2 ml, PCR-plate, 96-well Multiply--Strips

Cat. No. 1 667 041 N80-0560 N80-0933 0030127.307 0030124.200 0030127.374 72.985

Type 1 b b b b b

Type 2

b b

Note: If you use a PCR-plate which is not listed in the table above, first run a Post Elution protocol using an empty plate and select PCR-plate, Type 1. Perform one pipetting step into a well located in the center of the plate. If the instrument gets stalled during this operation ('error 010') because the Reaction Tips hit the bottom of the plate, you have to use this plate as Type 2. Important: Any PCR-plate that is used should not be vaulted
*) MicroAmp is a registered trademark of Applied Biosystems, Foster City, CA, USA.

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Select the appropriate cooling block by clicking on its

name. You can select a cooling block for both the left and right half of Cooling Unit 2. A preview of the cooling blocks placed in Cooling Unit 2 is displayed in the right half of the window:

Confirm selection of cooling blocks by clicking the

'OK' button or by pressing <Return>/<Enter>. Note: You can select and confirm the selection in one step by double-clicking on the respective cooling block name. <
Note: Although the MagNA Pure LC A-Ring Cooling

Block physically occupies the complete Cooling Unit 2 it is selected from the 'Small Cooling Block' menu. The left and right half of the cooling block, corresponding to A-Ring A and A-Ring B, can be selected individually as a Small Cooling Block.

In case, you have selected the A-Ring Cooling Block,

you are prompted to enter the A-Ring barcode(s) using a barcode reader.

After a cooling block has been selected the 'Define

Name / Set Volume' window opens automatically.

Use the 'Large Cooling Block' menu to select a large

MagNA Pure LC Cooling Block. Large cooling blocks occupy the complete Cooling Unit 2. Note: The only available large cooling block is currently the MagNA Pure LC Cooling Block, LC Sample Carousel (Cat. No. 2 189 704).

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7.3.2 Defining Type, Name, and Initial Volume of Cooling Block Positions

After having selected the MagNA Pure LC Cooling Block used for Post Elution pipetting you have to define type, name, and volume of used reaction vessel positions within the cooling block.
The Define Name/Set Volume window opens auto-

Position: Displays the label of the respective

matically after cooling block selection when programming a new Post Elution protocol or when loading an existing one. Besides, you may open the Define Name/Set Volume window at any time from the 'Actions' menu by choosing the option 'Define Name / Set Volume'.

The Define Name / Set Volume window resembles a

reaction or reagent vessel position: LC: LightCycler capillary position (only for the LC Sample Carousel and the LC Centrifuge Adapters Cooling Block) MM = Master mix position (only for the LC Sample Carousel and the LC Centrifuge Adapters Cooling Block) Tube = Reagent tube position (only for the Reaction Tubes Cooling Block) A1 to H12: PCR plate well position (only for 96-well PCR Plate Cooling Block) Ring: Reaction Tube within a COBAS Amplicor A-ring (only for A-Ring Cooling Block) MMLarge, MMSMall: Small and large reaction tube position within A-Ring Cooling Block

spreadsheet table in appearance. Fields which can be modified by the user are white, while informative fields filled by the software are displayed in grey.

For every cooling block position in which you place a

reaction vessel containing reagent or a sample for PCR enter the necessary information into the column fields Type, Name, and InitVol: At first, you have to define the reaction vessel type:

Type: A cooling block position

can be defined to contain a reagent, a sample/standard, or a control. After clicking on the small 'downward-arrow' at the right side of the entry field a pull-down menu opens. Select 'Reagent' , 'Samp./Std.', or 'Control'. The following table columns are filled by default and cannot modified by the user:

Block Name: Displays the name of the selected

cooling block (LC Carousel, LC Cooling Block, Reaction Tubes, PCR Plate, Cooling Block A-Ring)

L/R: Indicates the position (left/right) of the coo-

Defining a cooling block position as reagent or sample is especially important for transfer of capillary information to the LightCycler SAM-file which can be saved from the Post Elution Result Screen (see Chapter B, section 7.8 for details how to generate a SAM-file):

ling block in Cooling Unit 2 (only valid for small cooling blocks)

If you defined a cooling block position as 'Samp./ Std.' and you program to dispense from this position into a capillary the name defined for this position is transferred into the 'Name' field of the LightCycler SAM-file. continued on next page

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There are several options how sample names are forwarded to LightCycler capillary name fields of the SAM-file: a) No name is entered for a position defined as 'Samp./Std.': If a volume from this vessel is dispensed into a capillary, the capillary sample name remains empty, too. In the Post Elution-Result-Screen at the end of protocol execution, the user may enter a desired name, which will appear in the SAM-file. b) A Sample/Standard position is named (e.g., 'S1'): If a capillary receives a volume from this vessel, its sample name in the Post Elution Result Screen and the SAM-file will be 'S1', too. c) If a capillary receives two volumes from two different named Samples/Standard positions (e.g., 'S1' and 'S2') its sample name will be the combination of both Samples/Standard names (= 'S1+S2').

InitVol: Double-click on the entry field to enter the

initial (starting) volume of a reaction vessel.


Note: It is possible to define empty vessels (0 l

volume), e.g., vessels which are used for the preparation of dilution series or master mixes. If you define an empty vessel as 'Samp./Std.', but dispense volumes from two 'Reagent' type positions into that vessel, its type will change back to 'Reagent'. In that case, to keep 'Samp./Std.', one of the 'Aspirate From' positions has to be defined as 'Samp./Std.'.
Important: It is not sufficient to enter solely a volume

which corresponds to the Dispensing Volume multiplicated by the number of 'Dispense To' steps (e.g., if you want to dispense 15l of master mix into 32 capillaries this would correspond intrinsically to a total needed volume of 480 l). Each vessel from which liquid is aspirated must contain an Overdraw Volume in addition to the total volume dispensed into further vessels. This Overdraw Volume is calculated individually for each vessel depending on the actual Post Elution Protocol. It is recalculated after each change of the protocol. The overdraw volume is the sum of the dead volume of the reaction vessel, and liquid loss due to adhesion to the walls of tips and vessels as well as evaporation. Use the following tables as a lead for calculation of a sufficient initial volume, depending on the volume that is to be dispensed, and the number of 'Dispense To' steps. When performing 1:n multidispensing and tip discard after every pipetting step is activated, the required volume is larger compared to multidispensing with tip discard performed only after the last pipetting step. continued on next page

If you defined a cooling block position as 'Reagent' and you program to dispense from this position into a capillary the name defined for this position is transferred into the 'Comment' field of the LightCycler SAM-file (see Chapter B, section 7.8 for details how to genrate a SAM-file). Only after you have defined the type, the entry fields for name and initial volume will become accessible:

Name: Double-click into the entry field to enter the

desired name of the respective reagent or sample.


Note: The allowed length of position names is

25 characters. If you enter a name for a position defined as 'Reagent' and then change its type to 'Samp./ Std.', or vice versa, you have to re-enter the name.

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without tip discard 5 10 15 pipetting volume [l] 20 30 40 50 60 70 80 90 100 1 10 15 20 26 36 46 57 67 77 88 98 109 2 16 26 37 47 68 88 109 130 151 172 193 214 4 28 48 69 90 131 172 214 256 297 339 381 423 8 51 93 134 175 258 341 424 507 591 675 758 842

Number of pipetting steps 12 75 137 199 261 385 510 634 759 885 1010 1136 1262 16 98 181 264 347 512 678 845 1012 1179 1346 20 122 225 329 432 640 847 1055 1264 1473 24 146 270 394 518 767 1016 1266 32 193 359 524 690 1022 1355 64 383 715 1048 1380 96 575 1074

with tip discard 5 10 15 pipetting volume [l] 20 30 40 50 60 70 80 90 100 1 10 15 20 26 36 46 57 67 77 88 98 109 2 16 26 37 47 68 88 109 130 151 172 193 214 4 28 48 69 90 132 174 215 257 299 341 383 425 8 52 94 136 178 262 346 430 514 599 683 768 853

Number of pipetting steps 12 77 140 203 266 393 520 647 775 903 1031 1159 1287 16 102 187 272 357 527 697 868 1040 1211 1383 20 127 234 341 448 662 877 1093 1308 24 153 282 411 541 800 1060 1320 32 205 380 556 731 1082 1434 64 433 805 1177 96 689 1283

The MagNA Pure LC software also calculates the loss of liquid due to evaporation from the reaction vessel. Depending on the time period between placing a reaction vessel into the Cooling Block and dispensing the programmed volume of liquid from the vessel, additional liquid might be lost due to evaporation. Therefore, a larger initial volume will be needed to compensate for volume loss. Add additional 1 l for every 10 min of standing time.
It is absolutely necessary that the correct initial volume is actually present in the reaction vessel. The MagNA Pure LC Instrument does not check the correct liquid level in the reaction vessel, but relies on the settings made by the user.

To ensure pipetting of correct volumes it is important that all reagents pre-pipetted manually into reagent tubes are centrifuged to be free of air bubbles and that the reagents are equilibrated to cooling block temperature (temperature equilibration needs approx. 10 min,when using reagents with room temperature, or 20 min, when using reagents previously stored on ice, respectively). Only use calibrated pipettes for manual pre-pipetting.

Confirm each entry by either clicking the OK button, by pressing the <Return> or <Enter> key, or by pressing the <Cursor up> or <Cursor down> key. continued on next page

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If you want to print a list of all specified cooling block

positions, click the Print button. This printout may also be used as a pipetting scheme for filling the reagents used in the Coo-ling Block.

Click the 'Print' button for printing a list of the Cooling Block positions.

After a cooling block has been selected, and name and

initial volume of cooling block positions have been defined, you can start programming the actual Post Elution protocol steps.

7.3.3 Programming Post Elution Pipetting Steps

The MagNA Pure LC Instrument can perform two different kinds of pipetting actions during a Post Elution run: A defined volume is aspirated from a reaction vessel and dispensed into another reaction vessel. This action is programmed as Aspirate-Dispense set. A liquid volume inside a reaction vessel is mixed. This action is programmed as Mixing Step. The following sections describe all programming steps which are necessary to create a Post Elution Protocol.

Programming an Aspirate-Dispense Set: Aspirate from one vessel dispense into one vessel (1:1 single-dispensing)
Select the reaction vessel from which you want to aspi-

rate a volume by clicking on its position in the Post Elution-Steps Screen. The position will be labeled by a red ring:

displaying the position name. This may help you to identify the correct vessel:

After you selected the reaction vessel, the 'Aspirate

From' button is accessible. Click on it to define the Aspirate From position.

Important: Be sure that no other than the desired

position is selected and marked by a red ring. Otherwise you would include this additional position inadvertently into your Aspirate-Dispense set. When you move the mouse pointer over a reaction vessel position, a yellow information flag appears

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After clicking on the 'Aspirate From' button, the Dis-

Select the reaction vessel into which the aspirated

pensing Volume can be entered into the respective entry field. The entry field is filled automatically with a default value of 5 l. The position from which is aspirated is now labeled by a blue 'upward' arrow.
Note: Aspirating Volume and Dispensing Volume are

volume is to be dispensed by clicking on its position in the Post Elution-Steps Screen. The position will be labeled by a red ring and the 'Dispense To' button will become accessible. Click the button to confirm the selection.

identical. The 'Aspirating Volume' entry field is not active in this software version. If you select a position for which no InitVol was defined, the 'Define Vessel' window opens automatically. You can now enter the InitVol (see Chapter B, section 7.3.2 for details).

After clicking the 'Dispense To' button, the selected

position will be labeled by a red 'downward' arrow. The 'DONE' button is now accessible to confirm programming of the complete Aspirate-Dispense set.

Important: It is not possible to aspirate a larger volu-

me than 100 l. Aspirate-Dispense pipetting steps are performed using only small (yellow) Reaction Tips, whose capacity is maximally 100 l. If you enter a volume larger than 100 l into the 'Dispensing Volume' entry field, it will be marked red and you have to change it to an allowed value. Otherwise you will not be able to continue programming.

Important: 'Dispense To' positions are controlled by

If you want to dispense a volume greater than 100 l into a reaction vessel you have to divide it into several pipetting steps of maximally 100 l (e.g., dispensing of 250 l is programmed as two dispensing steps of 100 l and one of 50 l). It is strictly recommended not to enter Dispensing Volumes smaller than 5 l. The minimum volume which can be pipetted accurately by the instrument is 5 l. In addition, the Dispensing Volume must be an integer.

the software regarding vessel capacity. You are only allowed to dispense a limited total liquid volume into a reaction vessel. If this maximum vessel capacity is exceeded, the respective Aspirate-Dispense set will be marked as failed in the Protocol Description Table (i.e. the status line is color coded red):

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If you position the mouse pointer over the affected 'Dispense To' position a red information flag appears, informing you on the volume error.

After clicking the 'DONE' button the steps of the new-

ly programmed Aspirate-Dispense set appear in the Protocol Description Table:

If you want to verify the Aspirate-Dispense set, select

The volume capacities of the various reaction vessels that can be used during Post Elution are:
LightCycler capillaries: 50 l Wells of 96-well PCR-plates: 200 l Reaction tubes: 1500 l If you want to discard the Reaction Tip after the pipet-

it in the Protocol Description Table by clicking on the appropriate line numbers. The corresponding Aspirate-Dispense set will be highlighted black and you can see the corresponding "Aspirate From' and "Dispense To" positions.

ting step was performed mark the field 'Discard Tips'.


Note: The 'Discard Tips' option is always active by de-

fault. If you deactivate the function, it will automatically be activated again when you program the next Aspirate-Dispense Set.
If you want to halt the Post Elution run after comple-

tion of the current Aspirate-Dispense set, mark the field 'Pause'. The status line of the Aspirate-Dispense set will be color coded white. You may choose this function to place or exchange a reagent or disposable plastic (e.g., additional Reaction Tips) on the Reagent/Sample Stage after completion of a defined Aspirate-Dispense set. For instance, if you want to perform RT-PCR reactions, you first may use the Post Elution function to setup the Reverse Transcription (RT) reactions. After pipetting all reagents and samples for RT, you halt the Post Elution run by programming a PAUSE step, and put the reaction vessels into a suitable heat block for performing the RT reaction. After completion of the RT reaction, you put the reactions vessels back into the MagNA Pure LC Instrument and continue the Post Elution run for setup of PCR reactions.

Note: During programming of Aspirate-Dispense Sets

it is possible to use the keyboard instead of confirming the single pipetting steps by clicking the "Aspirate From" or "Dispense To" buttons:

Select the vessel from which is to be aspirated Press <Return> or <Space> Select the vessel into which is to be dispensed Press <Return> or <Space> Check dispensing volume Press <Return> or <Space> Press <Return> or <Space>

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Programming an Aspirate-Dispense Set: Aspirate from one vessel dispense into several vessels (1: n multi-dispensing)

If the same volume of one reagent (e.g., PCR master mix or water for dilution series) is to be pipetted into several different reaction vessels, it is more timesaving to program this as a 1:n multi-dispensing step than as several individual 1:1 single-dispensing steps. This is done by selecting all reaction vessels into which the liquid volume should be dispensed simultaneously.

Note: Although programming of the dispense steps is done

in one step, the actual pipetting steps are performed individually. It is not possible to aspirate the complete volume needed for several sequential pipetting steps and to dispense it successively.

Select the Aspirate From position and specify the

All "Dispense To" positions are now labeled by a red

Dispensing Volume as described in this section under Programming an Aspirate-Dispense Set: Aspirate from one vessel - dispense into one vessel (1:1 single-dispensing)
To select all reaction vessels into which the aspirated

'downward' arrow.

volume is to be dispensed select the first vessel, and then by holding the <Shift> key, click on the last reaction vessel. All vessels between the first and last one will be selected automatically. You can still add or remove individual vessels to or from the sequence by single mouse clicking. In the example shown, 15 l of a PCR mastermix are dispensed into 32 LightCycler capillaries. The positions will be labeled by a red ring and the 'Dispense To' button becomes accessible. Click on the button to confirm your selection:

Confirm programming of the 1:n multi-dispensing

step by clicking the 'DONE' button. In the Protocol Description Table all 32 pipetting steps are displayed as part of one Aspirate-Dispense set:

Select first vessel

hold Shift key select last vessel


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If the InitVol defined under "Define Name/Set Volume" is not sufficient to perform all programmed pipetting steps, the respective Aspirate-Dispense Set will be labeled as failed (the status line is displayed in red). In addition, the status line at the bottom of the screen displays the notification 'Volume error will occur in Row= '.

3. Choose the option Define Vessel. A settings window opens:

If you move the mouse-pointer over the affected "Aspirate From" position, a red information flag appears displaying volume information:

You can now define the type of the vessel, its name, and its initial volume. Define the type of the reaction vessel by clicking on Reagent, Sample/Std., or Control.
Note: Defining a reaction vessel as Control is es-

pecially useful when setting up reactions for COBAS Amplicor tests in the A-Ring Block.

Needed Vol: The additional needed volume to perform all programmed pipetting steps Needed Initial: The initial volume (InitVol) that has to be set by the user and pre-pipetted into the reaction vessel to allow performance of all programmed pipetting steps

Enter the new initial volume into the 'Volume' entry field. If an InitVol was already specified, the current value is displayed. Confirm your entry by clicking 'OK' or pressing <Return>/<Enter>. Enter the name into the Name entry field.
Note: If several vessels are selected at the same

time, all will be assigned to the same type and the same InitVol. The Name entry field is not accesible in that case.
Behaviour of the 'Discard Tips' function is different

Adjust the initial volume of the "Aspirate From" position to the correct volume and make sure that enough liquid is present in the reaction vessel. To change the InitVol either use the "Define Name/Set Volume" function or change it directly using a pop-up menu: 1. Select the reaction vessel whose InitVol you want to change by clicking on its position in the Post Elution-Steps Screen. You may select several individual vessels at a time by clicking on their respective positions, or a range of positions by clicking on the first and last position while holding the <Shift> key. The selected positions are labeled by a red ring. 2. Right-click on the selected reaction vessel positions. A pop-up menu appears:

for 1:n multi-dispensing sets compared to conventional 1:1 single-dispensing steps. The following two tip discard options are possible for a 1:n multi-dispensing set: The reaction tip should be discarded after completion of every individual pipetting step
mark the 'Discard Tips' field The status line of the Aspirate-Dispense Set

displays 'Discard=Yes'

The reaction tip shall be discarded only after the last pipetting step of the 1:n multi-dispensing set
do not mark the 'Discard Tips' field To emphasize the difference in Tip Discard

behaviour the status line of the AspirateDispense Set displays 'Discard=Afterwards'

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Programming an Aspirate-Dispense Set: Aspirate from several vessel dispense into several vessels (1:1 multi-dispensing)

Usually aspirate-dispense operations are performed in single steps using one Reaction Tip attached to the first nozzle (position A) of the nozzle head. But as the nozzle head offers 8 nozzles for Reaction Tips, it is also possible to perform up to eight aspirate-dispense operations in parallel, which will speed up the Post Elution process. As a pre-requisite for this type of pipetting operation the number of 'Aspirate From' and 'Dispense To' positions needs to be identical, and the reaction vessel positions have to be arranged horizontally in sequence. Furthermore, 1:1 multi-dispensing is only possible between positions of the Storage Cartridge, the LC Centrifuge Adapters Cooling
Select the reaction vessels

Block, and the 96-well PCR Plate Cooling Block. This is because the horizontal distance between the wells of these reaction vessels is identical to the spacing of the pipette nozzles. The Reaction Tube Cooling Block and the LC Sample Carousel Cooling Block cannot be used for 1:1 multi-dispensing. One application for this kind of pipetting operation is the transfer of eluted nucleic acid samples from the wells of the Sample Cartridge to LightCycler capillaries placed in the LC Centrifuge Adapters Cooling Block or wells of a 96-well PCR plate.

Select the same number of

from which you want to aspirate: Click on the first vessel position, and then by holding the <Shift> key on the last vessel position. All positions between the first and the last position are selected automatically. The positions are labeled by a red ring.

'Dispense To' positions, again by clicking on the first position and then by holding the <Shift> key on the last position.

After entering the Dispen-

After confirming the selection by clicking on 'Dispen-

sing Volume into the respective entry field and clicking on the 'Aspirate From' button, the positions from which the volume is aspirated are labeled by blue 'upward' arrows:

se To' and 'DONE' the 1:1 multi-dispensing set appears in the Protocol Description Table:

In contrast to 1:n Multi-Dispensing the 'Aspirate From' and 'Dispense To' positions are not listed individually in the Protocol Description Table but as a position range (e.g., A1-H1 for Storage Cartridge positions, or LC1-LC8 for LightCycler capillary positions).

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Programming a Mixing Step If, for instance, you want to prepare a dilution series or a PCR master mix using the Post Elution abilities of the
Select the reaction vessel

MagNA Pure LC Instrument, it is necessary to mix the content of a reaction vessel before further dispensing.
The Mixing Step appears in the Protocol Description

whose content you want to mix by left-clicking on it. The position is labeled by a red circle. Click on the 'Mix' button:

Table. To flag this programming step as Mixing Step the status line is color coded in yellow:

After clicking the 'Mix'

button you are prompted to enter how often you want to perform the mixing step into the 'Mixing Cycles' entry field:
Note: Maximum possible

Note: The dispensing volume for a mixing step is cal-

number of mixing steps is 15. To ensure proper mixing it is recommended to mix at least ten times.

culated automatically by the software. Up to a volume of 109 l inside the vessel small Reaction Tips are used for mixing. Above 109 l large Reaction Tips are used. The dispensing volume is calculated as 'total volume minus dead volume' (in case of Reaction Tubes the dead volume is 9 l when using small Reactions Tips, and 10-16 l when using large Reaction Tips, depending on the total volume). It is not possible to mix the volume within LightCycler capillaries.

After entering the

'Mixing Cycles' click on the 'DONE' button to confirm programming of the mixing step.
Note: After a Mixing Step

the Reaction Tip is automatically discarded. Therefore, the 'Discard Tips' check box is not accessible when programming a Mixing Step.

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The following passage shows an example for programming of a 10 fold dilution series:
Define Name/Set Volume Program the first dilution step (1:10 dilution):

Define seven reaction vessel positions: One position for the diluent (water) defined as 'Reagent' type One position for the undiluted DNA stock solution defined as ' Samp./Std.' Five positions for reaction vessels in which the dilution series is prepared by serially diluting 10 l of DNA with 90 l of water (to transfer the position name to the LightCycler SAM-file define them as 'Samp./Std. if you pipett from these positions further into LC capillaries)

Aspirate 10 l from the reaction tube containing the DNA stock solution Dispense it into the reaction tube scheduled for the 1:10 dilution Activate the 'Discard Tips' function, to avoid any cross-contamination Mix the content of the first dilution tube ten times. Mixing volume will be 91 l (100 l is the actual volume inside the tube, 9 l is the dead volume of a 1.5ml reaction tube)

Complete the protocol by programming the following Pre-dispense water into the reaction tubes used for

dilution steps:

dilution of DNA:

dispense 90 l of water into every reaction tube scheduled for dilution of DNA program as 1:n multi-dispensing step by selecting all dilution tubes as 'Dispense To' positions at once because you want to dispense one reagent several times it is sufficient to discard the used reaction tip only after the last dispensing step: Unmark the 'Discard Tips' check box.

according to the previous programming step program the Aspirate-Dispense Sets for the 1:100 to 1:100000 dilutions

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7.4

Saving and Loading a Post Elution Protocol 7.4.2 Loading If you want to load a previously saved Post Elution Protocol file from the File menu choose the option 'Load Post Elution'. The file selection window opens. The Post Elution protocol directory within the directory 'MagNA Pure' is opened automatically. Select the Post Elution protocol (*.pep) file you want to load and confirm your selection by clicking the 'Open' button.

7.4.1 Saving After you have programmed a new Post Elution protocol or modified an existing one, you can save the Post Elution Protocol file (*.pep) for further use.

From the File menu choose the option 'Save Post Elution'. The file selection window opens. The 'PostElution' directory within the directory 'MagNA Pure' is opened automatically:

Define a name and confirm saving of the Post Elution protocol (*.pep) file by clicking the 'Save' button.
Note: Up to MagNA Pure LC software version 2.11 infor-

mation of the Post Elution protocol is stored in two separate files: *.pep and *.pes. While the pep-file contains information on used Cooling Blocks and Cooling Block positions, the pes-file contains information on the actual Post Elution pipetting steps. Therefore, if you want to distribute a Post Elution protocol programmed with a software version previous to 3.0, always copy both file types. With MagNA Pure LC software version 3.0 Post Elution protocols are saved as single file (*.pep). If you load a Post Elution protocol file created with software version 2.1 into software version 3.0 it will automatically be converted into the new format. To keep the new format you have to save the protocol file. It is possible to load a Post Elution protocol which was created by a different Standard User. To prevent changes to another users protocol, changes made by another user can only be saved under a different file name.
Important: In the Post Elution protocol all parameters and

If a newly created or modified Post Elution protocol, not saved already, is still displayed in the Protocol-Description Table and you want to load a Post Elution protocol file, the following warning message appears:

Click on 'Yes' if you want to save the changed Post Elution protocol. Click on 'No' if you directly want to load a new Post Elution protocol file. Any unsaved protocol data will be lost. Note: If you load a Post Elution protocol which in-cludes the MagNA Pure LC Cooling Block, A-Ring, you are prompted to enter the barcode(s) of the COBAS Amplicor A-Ring(s) used.

settings of the reaction vessel positions and Aspirate-Dispense sets are saved. Also information on the samples, which are present within the Sample Cartridge during programming of the protocol are saved. Therefore, if loading the Post Elution protocol the next time, be sure that actual and saved sample data are matching to ensure correct processing of the Post Elution protocol.

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7.5

Editing a Post Elution Protocol

After you have programmed a new Post Elution protocol or have loaded an existing one, you may modify individual steps of the protocol displayed in the Protocol-Description Table may be modified by using built-in editing functions of the Post Elution Steps screen. Use the editing functions to:

Change the parameters of an individual AspirateDispense set ('Aspirate From' or 'Dispense To' position, tip discard status, dispensing volume etc.) Cut or copy one or several Aspirate-Dispense sets and paste them into a new position Delete one or more programming steps Insert blank rows into the Post Elution protocol Change names and/or initial volumes Delete reaction vessels

7.5.1 Editing the Protocol-Description Table To edit the programming steps shown in the Protocol Description Table you have to select one or several AspirateDispense sets:
Left-click on one of the line numbers belonging to the If you want to select a contigous range of Aspirate-

Aspirate-Dispense set you want to select


The Aspirate-Dispense set is highlighted black:

Dispense sets, hold the <Shift> key during clicking on line numbers belonging to different Aspirate-Dispense sets or drag the mouse-pointer while holding the left mouse button over a range of line numbers:

Important: Before programming the next regular

The settings currently active are shown: 'Aspirate From' and 'Dispense To' position, Dispensing Volume, Tip Discard status, Pause status.

Aspirate-Dispense set, which should be attached to the end of the protocol, always deselect any other set. If this is not done, the settings of all selected sets will be changed. To deselect, click on the large rectangle in the upper left corner of the Protocol Description table:

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Changing the parameters of an Aspirate-Dispense set


Select the Aspirate-Dispense set you want to modify

according to the previous instructions.


To change the Dispensing Volume enter a new value

into the 'Dispensing Volume entry field. To change the 'Tip Discard' or 'Pause' status mark or unmark the respective check-box. Confirm your setting by pressing the 'DONE' button
To change the 'Aspirate From' or 'Dispense To' po-

The 'Aspirate From' and the 'Dispense To' buttons become accessible. If you click on the 'Aspirate From' button, the selected position is defined as new 'Aspirate From' position. If you click on the 'Dispense To' button, the selected position is defined as new 'Dispense To' position. Confirm the change by clicking the 'DONE' button.
You may use this function to remove pipetting steps

sition, select the new position by clicking on it:

from a 1:n multi-dispensing set. Select the 1:n multi-dispensing set in the Protocol Description Table. All 'Dispense To' positons are labeled by a red 'downward'-arrow. Click on those positions which you do not want to use anymore in the multi-dispensing step. You may also use the <Shift>key function to select a range of positions. They are labeled by a red ring. Click on the 'Dispense To' and then on the 'DONE' button. The red 'downward'-arrow will disappear from the previously selected positions and they are no more part of the 1:n multi-dispensing set.

Deleting programming steps

Select the Aspirate-Dispense set(s) you want to dele-

te according to the previous instructions.


After having selected set right-click on one of the ac-

cording line numbers. A pop-up menu offering several editing function appears. Choose the function 'Delete Row(s). The selected Aspirate-Dispense sets are deleted from the Protocol Description Table.

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Using the Copy-Paste function


Select the Aspirate-Dispense set you want to copy After clicking on the 'Paste' or 'Paste After' menu item,

according to the previous instructions.


Right-click on one of the rows belonging to the selec-

the previously copied Aspirate-Dispense set is inserted before or after the selected set.
Note:

ted set. A pop-up menu offering several editing functions appears:

Choose the function 'Copy'. The selected Aspirate-

You may use this function if you want to program a set of identical Aspirate-Dispense sets which are to be performed sequentially. E.g., if you want to dispense a volume larger than 100 l, you have to program this in several steps with a maximal size of 100 l each. If, for instance, you want to dispense 400 l, program the step for dispensing 100 l just once. Copy this Aspirate-Dispense set and paste it three times after the initial 100 l AspirateDispense set. If you choose 'Cut' instead of 'Copy', the selected Aspirate-Dispense set is copied to the clipboard memory, but deleted from the original position at the same time.

Dispense set will be transferred to the clipboard memory.


Select the Aspirate-Dispense set before or after that

you want to paste the copied set. Right-click on a line number to get access to the edit pop-up menu. The functions 'Paste' or 'Paste After' are now accessible.

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Inserting blank rows into a Post Elution protocol If you want to program a new Aspirate-Dispense set within an existing Post Elution protocol, you have to create a new empty row first.
Select the Aspirate-Dispense set from the Protocol To insert a new Aspirate-Dispense at the position of

Description table before or after that you want to insert a blank row.
Open the edit pop-up menu by right-clicking on one

the blank row, select the row and perform programming as outlined in detail in Chapter B, section 7.5.1.

of the rows belonging to the selected set. Choose 'Insert a Blank Row' or 'Insert a Blank Row After'. According to the chosen function a blank row will appear before or after the selected Aspirate-Dispense set:

After confirming the Aspirate-Dispense set by clicking the 'DONE' button, the new set replaces the previously inserted blank row.

7.5.2 Editing reaction vessel parameters

If you want to modify reaction vessel specific parameters, like name, type, or initial volume, a reaction vessel specific edit menu can be used. At first the reaction vessel positions whose parameters you want to modify, has to be selected by left-clicking on it. You may select several individual vessels at a time by clicking on their respective positions, or a range of positions by clicking on the first and last position while holding the <Shift> key. The selected positions are labeled by a red ring. Then rightclick on the selected position. A pop-up menu appears offering two editing functions:

Define Vessel ... to change type, name, and/or initial volume of reaction vessel positions.

This function is described in detail in section 'Programming an Aspirate-Dispense Set: Aspirate from one vessel dispense into several vessels (1:n single-dispensing)'.

Remove Vessel to completely remove a reaction vessel from the Cooling Block

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Changing parameters a reaction vessel position


If you want to define or change the type of a selected

reaction vessel, its name, or its initial volume choose the option 'Define Vessel':

You can now define the type of the vessel, its name, and its initial volume.

Define the type of the reaction vessel by clicking on Reagent, Sample/Std., or Control.
Note: Defining a reaction vessel as Control is especi-

ally useful when setting up reactions for COBAS Amplicor tests in the A-Ring Block.

A settings window opens:

Enter the new initial volume into the 'Volume' entry field. If an InitVol was already specified, the current value is displayed. Confirm your entry by clicking 'OK' or pressing <Return>/<Enter>. Enter the name into the Name entry field.
Note: If several vessels are selected at the same time, all

will be assigned to the same type and the same InitVol. The Name entry field is not accesible in that case.

Removing a reaction vessel

If you want to remove a reaction vessel from a cooling block, choose 'Remove Vessel' from the edit pop-up menu.

If you want to delete the vessel confirm the message by clicking the 'OK' button or pressing <Return>/<Enter>.
Note: It is only possible to remove master mix positions of

a Cooling Block, but not LightCycler capillary positions or positions from a 96-well plate.
Important: Only the reaction vessel will be removed from

The following warning message will appear:

the cooling block. All Aspirate-Dispense sets from the active Post Elution protocol connected to this reaction vessel, will remain and are displayed as failed after removing the vessel.

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7.6

Starting a Post Elution run


If you want to stop the Post Elution run (e.g., to place

After you have created a new Post Elution protocol or

have loaded a previously saved file, from the Actions menu choose the option 'Start Post Elution':

an additional reagent or to fill up reaction tips) click on the 'Pause/Stop' button. The following window opens:

The following message window appears:

Press the 'OK' button, if you want to start the Post Elution run, or press 'Cancel' if you want to abort the process.
Important: To start a Post Elution run, it is necessary

You may now open the instrument's front door and perform manual handling steps. If you want to continue the run, click the 'Resume' button. If you want to stop the run completely, click the 'Stop' button.
Important:

that Cooling Unit 1 (Storage Cartridge) and Cooling Unit 2 (MagNA Pure LC Cooling Block) have reached their working temperature of 7.5C. Temperature status of both cooling units is displayed by two status indicators named 'Cool1' and 'Cool2' in the lower right corner of the Post Elution-Steps Screen. But even if the final temperatures of Heating and Cooling Unit are not reached yet you may start the Post Elution run. But the actual pipetting process will not start before the temperature is reached.
If the current Post Elution protocol was not yet saved,

Before you start a Post Elution run: Make sure that no obstacles are present on the Reagent/Sample Stage

To ensure pipetting of correct volumes, it is important that all reagents pipetted manually into reagent tubes are centrifuged to remove air bubbles and to ensure that the reagents have reached Cooling Block temperature (temperature equilibration needs approx. 10 min,when using reagents at room temperature, or 20 min, when using reagents previously stored on ice, respectively). To ensure correct pipetting, it is also necessary to use only 1.5ml Sarstedt reaction tubes with screw caps. Sometimes air bubbles might get trapped at the bottom of a Storage Cartridge well. To ensure that the complete defined sample volume is aspirated, always check the Storage Cartridge for air bubbles. If necessary remove air bubbles manually. Make sure that enough small Reaction Tips are present on the Reagent/Sample Stage.

a message window opens, prompting you to save the Post Elution file:

Click on 'Yes' if you want to save the changed Post Elution protocol. Click on 'No' if you directly want to load a new Post Elution protocol file. Any unsaved protocol data will be lost. During the Post Elution run currently processed Aspirate-Dispense sets are highlighted in the Protocol-Description table.

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7.7

Viewing the result on the Post Elution Result Screen


Note: If a series of Aspirate-Dispense steps of 'Samp./Std.'

After completion of the Post Elution run the Post Elution Result Screen opens automatically, showing sample data and result status of all positions into which a volume from a reaction vessel defined as 'Samp./Std.' was dispensed.

volumes was performed only the result of the 'Dispense To' position of the last step is displayed.

Menus and Graphical Elements of the Post Elution Result Screen


In this area of the Post Elution Result Screen, in addition to the selected Post Elution protocol as well as MagNA Pure LC and LightCycler kit information, the parameters of the purification protocol defined on the Sample Ordering Screen are shown. The data are identical to those shown on the Result Screen after completion of the purification run. Purification run specific information is only shown if the Post Elution run was initiated from a purification run Result Screen.

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Result Table
Pos: Name of the cooling block position (e.g., LC = capillary position of a LightCycler Cooling Block) Sample Name: Name of the 'Samp/Std.' position from which a volume was dispensed into this reaction vessel (name of the 'Samp./Std.' position is defined on the 'Define Name/Set Volume' window) Type: For LightCycler capillary positions a sample type can be defined and transferred to the LightCycler 'Edit Samples' window via SAM-file. Click on the downward-arrow field to open a drop-down menu offering several type options (unknown, standard, positive, negative) Replicate of: For LightCycler capillary positions a sample can be defined as replicate of another sample Note: Here the name of a 'Reagent' position from which a volume was dispensed into this reaction vessel appears Concentration: For LightCycler capillary positions defined as 'Standard' the concentration value of the standard template can be entered (transferred to the LightCycler 'Edit Samples' screen via the SAM-file) Template Vol: Volume that was dispensed from a 'Samp./Std.' vessel into this position Result: Displays the result status of the position Pass (green): The 'Dispense To' operation was performed correctly Fail (red): The 'Dispense To' operation failed Code: If the result of the position is flagged as 'Fail' the respective error number is shown in this column

File Menu
Generate SAM-file: Saves the result screen data as a LightCycler sample file (*.sam). This function is only accessible if sample volumes were dispensed into LightCycler capillary positions. Save Result: Saves the sample data displayed on the Post Elution Result Screen as file (*.per) for documentation purposes. Print Post Elution: Prints the Post Elution Result table. Close: Closes the Post Elution Result Screen and returns to the screen from which Post Elution handling was accessed.

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7.8

Generating a LightCycler SAM-file

If during Post Elution pipetting sample volumes were dispensed into LightCycler capillaries during Post Elution, capillary specific sample data of the Post Elution Result Screen can be saved into a LightCycler sample file (*.sam) Generate a SAM-file as follows:
In the File menu of the Post Elution Result Screen

and reloaded into the LightCycler 'Edit Samples' screen. LightCycler specific sample parameters like type, replicate of, and concentration can be specified directly in the Post Elution Result Screen.

choose the option the 'Generate SAM-file'.

If you click the 'Close' button before the SAM-file was

saved, the following warning message appears. You can save the SAM-file now. Hereafter the Post Elution Result Screen is closed.

Note: This function button is only accessible if sam-

ple volumes were dispensed into LightCycler capillary positions.


A window opens for selection of a file save destinati-

on. Choose an appropriate drive and folder. The name of the SAM-file is generated automatically by the software. Confirm your selection by clicking the 'OK' button or pressing <Return>/<Enter>.

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8.

A-Ring Workflow

MagNA Pure LC software version 3.0 offers an especially adapted software workflow to use the instrument as front-end sample preparation system in combination with COBAS AMPLICOR tests.
Important:

MagNA Pure LC system is intended for general laboratory use only. It was neither developed nor validated by the manufacturer for any kind of in-vitro diagnostic application. Any use of the MagNA Pure LC system as a front-end sample preparation system for in-vitro diagnostic test systems (e.g., COBAS AMPLICOR tests) is in the sole responsibility of the user. The validation of the combination MagNA Pure LC nucleic acid isolation and in-vitro diagnostic testing must be done by the user, following the relevant national rules. Thus, the A-Ring workflow regards only the software handling in case the MagNA Pure LC Instrument is used in combination with COBAS Amplicor A-Rings and the respective MagNA Pure LC Cooling Block. It does not include reagents, purification protocols, or application-related working instructions specific for COBAS AMPLICOR sample preparation.

The so called A-Ring workflow starts with a special sample ordering screen, the A-Ring Ordering Screen . Further and integral part of this workflow is the MagNA
Pure LC Cooling Block, A-Ring, which enables to automat-

Features of the A-Ring workflow comprising A-Ring Ordering Screen, Start Information Screen, Result Screen, and Post Elution handling are:

ically setup COBAS AMPLICOR PCR reactions by combining eluted samples and PCR reagents of COBAS AMPLICOR tests directly within the reaction tubes of COBAS AMPLICOR A-Rings by Post Elution pipetting. See Chapter A, section 3.2.1 for a detailed description of the MagNA Pure LC Cooling Block, A-Ring. Main purpose of the A-Ring workflow is to ensure proper sample tracking from the primary sample to COBAS AMPLICOR PCR reaction setup.

Demand to enter barcodes of primary samples during A-Ring Ordering Limitation of sample number to a maximum of 24 (corresponding to two complete COBAS AMPLICOR A-Rings) Possibility to create and print sample barcodes for nonbarcode labeled primary samples Obligatory printing and entering of Sample and Storage Cartridge Barcode. Sample Ordering and Result files can be saved and loaded by using this barcode as file name. This ensures unambigous identification and assignment of the individual samples to the cartridge. Obligatory entering of A-Ring Barcodes.

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The following flow chart gives a schematic overview over the A-Ring workflow:

Load the A-Ring Ordering Screen from the Standard User Main Menu Screen

Print Sample/Storage Cartridge barcode (= BatchID)

Read primary sample barcode

Optional, if using non-barcoded primary samples: Print primary sample barcode Save Sample Order file (*.sod): Use Sample Cartridge barcode as file name

Proceed to the Start Information Screen by clicking on the Stage Setup button

Print Sample Order

Read Sample Cartridge barcode

Use as Sample Cartridge pipetting scheme

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Optional, if a Post Elution protocol containing the A-Ring Block has been selected on the A-Ring Ordering Screen Read A-Ring barcode

After confirmation of correct Reagent/Sample Stage setup start the purification run by clicking the OK button

Print Result Screen: Lists BatchID = Sample Cartridge Barcode

Save Result Screen file (*.ird): Use Sample Cartridge barcode as file name

Proceed to the Post Elution Steps Screen. Create or load a Post Elution protocol using the A-Ring Cooling Block.

Optional, if you do not want to use A-Ring barcode: Read A-Ring barcode(s) Print Cool Block barcode (=BatchID)

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Save Post Elution Result Screen file (*.per): Use A-Ring or Cool Block barcode as file name

Post Elution Result Printout

Setup and start COBAS AMPLICOR run

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8.1

Overview over A-Ring Ordering Screen specific functions


The A-Ring Ordering Screen is used for input of sample and purification protocol parameters. Its principle usage is identical to the standard Sample Ordering Screen. Therefore, in the following only features and functions different to the Sample Ordering Screen are explained. For information on the general usage of the Sample Ordering Screen see Chapter B, section 3.

Menu Bar of the A-Ring Ordering Screen File Menu


All functions of the A-Ring Ordering File menu as well as those of the other menus are identical to the Sample Ordering Screen, except:

Print Sample Barcode Prints a Sample Barcode (if an optional barcode printer is installed). To print a barcode, select a sample line in the Sample Order Table. Note: Printing a Sample Barcode serves two functions: If you start from primary samples which are barcode labeled and you read-in the barcode into the Name field of the Sample Order Table, you may print this barcode again for labeling of storage vessels into which the eluate might be transferred. If you start from primary samples which are not barcode labeled you can use this function to create a barcode label from scratch. In this case, manually enter a valid barcode into the sample name field of the Sample Order Table. Select the respective sample line to print the barcode. Print Cartridge Barcode Generates and prints a barcode label (if an optional barcode printer is installed) which can be used to track and identify the Sample and Storage Cartridge of the current experiment. The Cartridge Barcode is created by the software and consists of the last three digits of the instruments serial number plus a batch number. The Cartridge Barcode is identical to the Batch ID which is also listed on the Result Screen printout, and the Post Elution Result Screen printout (if Post Elution is performed in combination with a prior purification run).

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The barcode can subsequently be read-in for naming of the Sample Order and Result Screen file (if an optional Barcode Reader is installed). When you want to re-load the Sample Order or Result Screen file, simply read-in the barcode from the Storage Cartridge. On a Start Information Screen initiated from an A-Ring Ordering Screen you are then prompted to enter a barcode for the Sample Cartridge which you can create beforehand using the Print Cartridge Barcode function.

Entry Fields of the Sample Ordering Screen


All fields and functions needed for entry of protocol and sample information (i.e. Protocol, Post Elution Protocol, Sample Volume, Elution Volume, Dilution Volume) are identical to the standard Sample Ordering Screen. In addition, the A-Ring Ordering Screen offers two fields for entry of COBAS AMPLICOR test specific data: Cobas Amplicor Kit name: Enter the name of the COBAS AMPLICOR Kit used here for documentation purposes.

Cobas Amplicor Kit lot: Enter the lot number of the COBAS AMPLICOR Kit used here for documentation purposes.

Graphical Elements of the Sample Ordering Screen


With exception of the Sample Order Table all graphical elements of the A-Ring Ordering Screen are identical to the standard Sample Ordering Screen. Sample Order Table The Sample Order Table of the Ordering Screen is restricted to a maximum number of 24 samples which corresponds to two complete COBAS AMPLICOR A-Rings. The Sample Order Table lists the samples and their data (name, type, position). In addition, it can directly be used for entry of sample data. #: Displays the sample identification number T: Displays the sample type. You can choose between C = Control, or S = Sample. To change the sample type click on the T field of the respective sample line. Sample Name: Displays the corresponding sample name or sample barcode. For A-Ring Ordering use only sample barcodes for sample identification. Sample Volume: Displays the sample volume. This value is automatically transferred from the Sample Volume entry field.

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8.2

A-Ring Ordering Workflow

On the Standard User Main Menu Screen click the

function button A-Ring Ordering.

The A-Ring Ordering Screen opens with a message

which reminds you to enter the sample names with a barcode reader:

After you have entered all samples select the appro-

priate purification protocol and specify the protocol parameters (Sample volume, Elution volume, Dilution Volume) as described in detail in Chapter B, section 3.2.
If you want to perform automated setup of the

COBAS Amplicor PCR reactions by Post Elution pipetting using the A-Ring Cooling Block in direct combination with the purification run, select an appropriate Post Elution protocol from the Post Elution_Protocol menu.

Using the Print Cartridge BarCode function from To enter sample names (bar codes) follow the descrip-

tion under Sample Ordering Screen Enter Sample Names. In brief, in the Sample Order Table click on the Sample name field of the first sample. Read in the sample barcode. Proceed to the next Sample Name field. Always start with position A1 and enter samples in a continous sequence.
If you start from non-barcoded primary samples you

the File menu, print a barcode label both for the Sample Cartridge and the Storage Cartridge. Paste the barcode label onto the provided area of the cartridges.

can use the Print Sample Barcode function to create barcode labels (if an optional barcode printer is installed). Enter a valid barcode manually into the sample name field of the Sample Order Table. Select the respective sample line and choose the function Print Sample Barcode from the File menu.

The Cartridge Barcode is created automatically by the software and consists of the last three digits of the instrument serial number plus a batch number. continued on next page

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The Cartridge Barcode is identical to the Batch Identification Number (BatchID) of the current purification run. The BatchID is also listed on the Result Screen printout as well as the Post Elution Result printout (if the Post Elution run is performed in combination with a prior purification run). To ensure correct loading of the samples into the Sample Cartridge, use the Print Sample Name function from the File menu to print out a loading list, which can be used as pipetting scheme:

If being opened from the A-Ring Ordering Screen the

Start Information Screen exhibits additional features:

During confirmation of the correct setup of the Reagent/Sample Stage the software prompts you for input of the barcode for the Sample Cartridge when you click on the Sample Cartridge text box. It is only possible to proceed if the correct cartridge barcode is read-in.

If a Post Elution protocol including the A-Ring Cooling Block has been selected on the A-Ring Ordering Screen, you are also prompted to enter the A-Ring barcode(s) (depending on whether you use one or two A-Rings), when you click on the cooling block text box.

After verification of all input parameters click on the

Stage Setup button to proceed to the Start Information Screen.

The software uses the sample information defined on the Sample Ordering Screen to calculate which disposable plastics and volumes of isolation reagents are needed. This information is displayed in form of the Stage Layout Graphic on the Start Information Screen. Use the Stage Layout Graphic as a guide to place the correct number of disposable plastics and the correct volume of reagents on the Reagent/Sample Stage (see Chapter B, section 4.1 for a detailed description of the Start Information Screen).

It is only possible to proceed if a correct A-Ring barcode is read in.


When confirmation of the Reagent/Sample Stage

setup is completed by clicking all text boxes, and if the Dispo Lockbar as well as the instrument cover are closed and locked (the respective status indicators display 'green'), the OK button becomes accessible. You can now start the purification run. continued on next page

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Purification Run

Progress of the purification run can be followed on the Batch Status Screen. After completion of the run the Result Screen appears Print out the Result Screen information. The printout lists the BatchID which is identical to the Cartridge Barcode. Save the Result file to create a permenant record of your experiment. As file name use the respective barcode from the Storage Cartridge. Sample specific information is now connected to a specific Storage Cartridge and barcode. If you want to reload the sample information at a later timepoint use the Load Result Screen function from the A-Ring Ordering Screen File menu. As file name read in the barcode from the Storage Cartridge and the previously saved Result file is unambigously identified.

Post Elution Handling

You can now proceed to the Post Elution Steps Screen for automated setup of the COBAS AMPLICOR PCR reactions by combining eluted nucleic acid samples and PCR reagents of COBAS AMPLICOR tests directly within the reaction tubes of COBAS AMPLICOR A-Rings by Post Elution pipetting. Create a new Post Elution protocol or load an existing one containing the MagNA Pure LC Cooling Block, A-Ring. See the section Post Elution Steps Screen for details how to programm Post Elution protocols. You will be prompted to enter the barcode(s) of the COBAS AMPLICOR A-Ring(s) used.

Alternatively, if you do not want to use the A-Ring barcode(s) you can create your own barcode(s) using the function Print Cool Block Barcode from the File menu. If performed in combination with a prior purification run, the Cool Block Barcode will be identical to the BatchID, with the figure 1 added for the left A-Ring Cooling Block part, and the figure 2 added for the right A-Ring Cooling Block part.

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After completion of the Post Elution run, the Post Elution Result Screen appears. The Result Table is divided into two parts to separate the positions of A-Ring A from A-Ring B.

Batch ID (= Cartridge Barcode = Cool Block Barcode)

A-Ring A A-Ring A

A-Ring B A-Ring B

Print out the Post Elution Result which lists the BatchID.

Save the Post Elution Result file (*.per) using either the A-Ring barcode(s) or the Cool Block barcode as file name.

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9.
9.1

Service functions
Liquid Waste Discard

After completion of a purification run used buffers and reagents are left over in the Processing Cartridges. Discard of these used buffers and reagents may be done manually or by an automatic function provided by the MagNA Pure LC Instrument, called Liquid Waste Discard. Use of this automatic discard function may be of advantage if e.g potential

ly infectious sample materials were used or possible contact to irritant compounds of the reagents is to be avoided. If you want to remove all used buffers and reagents from the Processing Cartridges into the Liquid Waste Bottle do the following:

Click the Liquid Waste Discard button on the Main Menu Screen.

To activate Liquid Waste Discard click the START

button. Liquid waste is then transferred from the Processing Cartridges into the Liquid Waste Bottle.
Important: No volume control for the Liquid Waste Discard Bottle exists. Therefore, when using this function you should always control the volume level within the Liquid Waste Bottle before starting the Liquid Waste Discard. Note: You may also perform Liquid Waste Discard automatically after completion of a purification run. To do this, activate the Liquid Waste Discard check box on the Sample Ordering Screen. See Chapter B, section 3 for details.

A window displaying a schematic overview over the

Processing Cartridge area of the Reagent/Sample Stage opens.

By mouse clicking select those Processing Cartridge

positions from which you want to discard liquid waste. Confirm placement of the Liquid Waste Discard Bottle, the Solid Waste Bag, and the Liquid Drop Catcher disposable by clicking on the respective text boxes.

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9.2

Decontamination
Important: Do not spray any reagent directly into the in-

The MagNA Pure LC Instrument includes a UV lamp (emission 260 nm) for decontamination of the Reagent/ Sample Stage from nucleic acids (see Chapter A, section 2.3). Operation of the UV lamp is regulated by the user via the MagNA Pure LC software. It is recommended to decontaminate the workstation from nucleic acids at least at the end of each work day. Areas of the workstation directly exposed to the UV light, like the center of the Reagent/Sample Stage are sufficiently decontaminated after 8 hrs. Areas further away from the UV light or located in the UV 'shadow' (e.g., the magnetic plate or corners of the instrument's housing) might not be sufficiently decontaminated after 8 hrs. Because of this, if assuming a severe contamination of the workstation with nucleic acids, wipe the incriminated instrument areas or parts additionally with a decontamination reagent (see Chapter B, section 2.3.1).
Click the 'Decontamination' button on the Main

strument, because electronic or optical devices might be damaged.


Note: Be aware that decontamination by using the inbuilt

UV light is not sufficient to destroy DNases/RNases or to disinfect the workstation after processing of infectious sample material:
For removal of DNases/RNases use LTK-008 or

RNase ZAP (see Chapter B, section 2.3.1 for details).


For disinfection use a suitable disinfection reagent

(e.g., Microzid or Kohrsolin ID) To decontaminate the inner part of the instrument after a run using the inbuilt UV lamp, do the following:

After start of the decontamination the Remaining

Menu Screen:

Decontamination Time is displayed.

The Decontamination Screen appears:

Note: The PC has to be on during the complete de-

contamination period. The recommended decontamination time is at least 8 hours.


If you want to stop decontamination before end of the Set the decontamination time by clicking the upward/

downward arrow to the right of the number fields.


From the Actions menu choose the option 'Start De-

contamination' to activate the UV lamp.

set period of time from the Actions menu choose the option 'Stop Decontamination' . You can also choose 'Close' from the file menu to stop decontamination and to leave the Decontamination Screen at the same time. Note: If you open the front door during active decontamination, the UV lamp is turned off automatically and the countdown is suspended until you close the door again.

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9.3

Exchanging Messages

Using the MagNA Pure LC software a user can leave a message for one or several other users, including the current user himself. When the addressed user logs in, the user is immediately notified of any new messages by the software.
9.3.1 Leaving a Message To leave a message, do the following:

One may use this function to draw the following users attention to any important incident concerning the instrument, e.g., a missing disposable or the possible risk of contamination.

On the Main Menu Screen click the 'Leave Message'

button

The Leave Message screen opens:

Select the user to whom you want to address the message.


Type the message you want to send to the selected user

into the Message field:

To send the message choose the option 'Send Message'

from the Actions menu:

To create a new message choose the option 'Create

Message' from the Actions menu:

A message window opens, asking you whether to send

the message or not. Click the 'OK' button or press <Return>/<Enter> if you want to send the message.

A new, empty message row is added to the Message

Table.

If you want to delete a message from the Message Click on the downward arrow button of the Username

field to see a drop-down list of all available user acounts:

Table choose the option 'Delete Message' from the Actions menu.

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9.3.2 Viewing a Message After log-in into the MagNA Pure LC software the additional function button 'Show Message' is displayed on the Main Menu Screen, if a message was posted for the respective user:

Click on the 'Show Message' button to view current messages. The 'Got Message' screen opens:

9.4

Installation of Cooling Blocks

If a new MagNA Pure LC Cooling Block for reaction setup using the Post Elution function is available, the module files containing its dimension parameters and the image files for graphical representation of the Cooling Block on the Post Elution Steps screen, have to be installed into the MagNA

Pure LC software before the Cooling Block can be selected and used. For installation of Cooling Block files an external software module is used: The Block Installer.

To install a new MagNA Pure LC Cooling Block do the following:


Log-in into Windows 2000 as User Administrator The Block Installer software starts. At first you will see

(ADMIN) (see Chapter B, section 2.2 for details)


Start the Block Installer software. In the Windows

a message window, reminding you to close all MagNA Pure LC related software. Follow this instruction and confirm by clicking 'OK'.

2000 task bar click on the Start button and choose 'Programs > MagNA Pure > Block Installer':

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In the Block Install Menu screen all currently instal-

To finally install the Cooling Block into the software

led Cooling Blocks are listed in the 'Current Block List'.

click the 'Install' button. You are prompted to confirm your action:

If a Cooling Block with identical name is already installed, the following warning message appears:

To install a new Cooling Block choose the function

If you want to deinstall a Cooling Block from the

'Select Block' from the File menu:

software, select its name in the 'Curent Block List' and choose the function 'Remove Block' from the Actions menu:

The file select window opens. Select the appropriate

Cooling Block file (*.mod) and confirm your selection by pressing 'open':
The following warning message will appear:

After confirming removal of the Cooling Block the

following information window opens.


After loading the Cooling Block module file, the

block parameters are displayed on the screen.

To avoid inadvertent deletion of Cooling Block files they are converted to backup files, which could be reverted and reloaded.

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9.5

Installation of Purification Protocols

If a new MagNA Pure LC Nucleic Acid Isolation Kit is available, an existing purification protocol was changed, or for an existing kit an additional purification protocol is available, the respective purification protocol has to be installed into the MagNA Pure LC software. To install purification protocols an external software module is used: The Protocol Installer. Each purification protocol consists of two file types: A blkfile and a plg-file. Only if a new isolation kit or a feature of To install a new Purification Protocol do the following:
Log-in into Windows 2000 as User Administrator

an existing kit is changed a new kit-file has to be installed in addition to the blk- and plg-file. As a pre-requisite for successful installation copy the files of one purification protocol onto a floppy disk (this is guaranteed when using protocol floppy discs available from Roche Applied Science; please contact your local Roche Applied Science representative).

To install a new kit and/or protocol first insert the flop-

(ADMIN) (see Chapter B, section 2.3.1 for details)


Start the Protocol Installer software. In the Windows

py disk on which the protocol/kit files are stored into the disk drive of the MagNA Pure LC PC. Then select the function 'Select Protocol' from the File menu:

2000 task bar click on the Start button and choose 'Programs > MagNA Pure > Protocol Installer':

The file selection window opens. Change the file path

to 3 Floppy (A:\ ) and select the protocol file to be installed:

The Protocol Install Menu screen appears. On the

right side of the screen currently installed reagent kits and purification protocols are listed in the 'Current Kit List' and the 'Current Protocol List', respectively:

After clicking the 'Open' button information on the

protocol to be installed is shown on the screen:

To complete the installation choose the function

'Install' from the Actions menu:

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The 'Save As' file selection window opens.

The newly installed purification protocol and the

Find the appropriate folder in which you want to install the new protocol files (e.g., 'mRNA HS') and double-click on its icon/name. Click on the 'Save' button to finish the installation process:

possibly newly installed kit are displayed in the 'Current Protocol List' and 'Current Kit List', respectively:

This step is only necessary if you want to install a new kit:

Set the file path to the directory 'FolderForProtocols' located in /Program Files/MagnaPure/Protocols/ and create a new folder inside the 'FolderForProtocols' directory with the name of the new kit:

To delete a protocol or a kit from the software select its name in the 'Current Protocol List' or 'Current Kit List, respectively, and choose the function 'Remove Protocol' or 'Remove Reag.Kit' from the Actions menu:

Click on the 'Create New Folder' button in the task bar of the file select window.

A new folder named 'Empty Folder' is created. Rename it according to the name of the reagent kit you want to install:

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9.6

Log File Backup

The MagNA Pure LC records all errors and distinctive features that occur during a purification run and a Post Elution run into several log files. These log files are:

MagNA Pure LC software log files

File

Content

Error.log

The last 1000 errors are registered together with a summary of instrument-specific data and the date at which each error occurred. Format of the Error.log file entries: Code: Error Code Date/Time: The time and date at which the error occurred Act.: The total operating time of MagNA Pure LC software Prep: The total number of samples treated. Version: The software version when the error occurred. Serial: The serial number of the MagNA Pure LC

bbiosx.log *)

The Bios system of the MagNA Pure LC software records into the bbios.log file when an error occurs. The Script commands are executed and the results are recorded.

MagNAx.log *) PELogx.log *)

The button handling and the screen changes of MagNA Pure LC software are recorded. The Post Elution pipetting steps are recorded.

*) Depending on the total processing time of the instrument several log files of the same type might exist. 'x' represents a running number that is added to the file name to differentiate multiple files of the same type.

If an error occurred during a purification or Post Elution run the log files contain relevant information for further troubleshooting. To ease sending of the log files to the Roche Molecular Biochemicals technical service, the MagNA Pure LC software offers a convenient way for backing up these files to a floppy disk. If you want to backup the log files to a floppy disk start the Log Backup program. In the Windows 2000 task bar click the 'Start' button. On top of the appearing program menu, you find the entry for the MagNA Pure LC Log Backup:

Click on the menu entry 'MagNAPure Log Backup' to start the program. A DOS program window opens:

You are prompted to insert a newly formatted floppy disk into drive A. Copy the log files onto the disk by pressing any key. After the copy process is completed you can leave the programm by pressing any key

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Chapter C

Appendix

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1.

List of Error Codes

When an error occurs, MagNA Pure LC software indicates the nature of the error by an Error Code Log Number. The syntax of the Error Code Log Number is:

000
Error Codes (Error Message Resource ID) Submodule Number Module Number Error Level W/E/F (Warning/Error/Fatal)

Meaning of the Error Code Log Number


The Error Code Log Number specifies the place the error occurred (Module and Submodule), the nature of the error (3-digit Error Code), and the severity of the error (Error Level).

Code 001

Level -

Label *)1 ERR_BIOS_INITIALIZE

Description Failed to Initialize BIOS

Possible cause Action Hardware problem:


instrument is not turned on no connection between PC and instrument program files not installed correctly T-NET PC board not installed correctly

Check the connection between PC and MagNA Pure LC Instrument. Call a service engineer. 002 ERR_IF_BIOS_NOT_EXEC Cannot execute SX BIOS.EXE Hardware problem:

no connection between PC and instrument program files not installed correctly

Check the connection between PC and MagNA Pure LC Instrument. Contact technical service. 003 ERR_IF_OPEN_MAPPING already File Mapping External Windows NT error Open Windows Task Manager by [Ctrl]+[Alt]+[Delete]. If BBIOS.exe and tMan.exe exist in the Process list, abort them. 004 ERR_IF_VIEW_MAP Cannot map I/F View External Windows NT error Open Windows Task Manager by [Ctrl]+[Alt]+[Delete]. If BBIOS.exe and tMan.exe exist in the Process list, abort them. 005 ERR_IF_OPEN_MUTEX Cannot open Mutex External Windows NT error Open Windows Task Manager by [Ctrl]+[Alt]+[Delete]. If BBIOS.exe and tMan.exe exist in the Process list, abort them.

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006

ERR_IF_INI_CMD_IF

Cannot make Process Interface

External Windows NT error Open Windows Task Manager by [Ctrl]+[Alt]+[Delete]. If BBIOS.exe and tMan.exe exist in the Process list, abort them.

007

ERR_SCR_INVALID_CMD

Invalid script command

Internal software error Contact technical service. The motor cannot move back to home position:

008

ERR_INI_MOTOR

Failed to initialize Motor

due to no motor movement. due to home position or limit sensor of the motor are broken.

Call a service engineer. 009 F ERR_MOTOR_BUSY Motor is busy The controller of a motor keeps working and cannot complete action:

due to no motor movement. because the home position sensor is broken The motor controller is hung-up. because T-Net PC board interface cannot setup communication. liquid drop catcher disposable not correctly positioned

Check correct position of Liquid Drop Catcher Disposable (see Chapter B, section 2.4 for details) Check the connection between PC and MagNA Pure LC. Turn off main power and turn on 10 minutes later. (MagNA Pure LC software also has to be rebooted.) Call a service engineer 010 W ERR_MOTOR_EM_STOP Motor stopped abruptly Hardware error:

the front door was opened during the run placement of disposables and/or accessories is not correct causing a nozzle head crash EasyTeaching was done at the wrong position wrong speed setting of the motor

Check Reagent/Sample Stage for correct placement of disposables and accessories or possible obstacles. Call a service engineer. 011 E ERR_MOTOR_RANGE_OVER Motor is beyond effective coordinates 012 E ERR_MOTOR_XY_LESS_HEIG HT 013 E ERR_MOTOR_NOT_BINARY Cannot move XY due to less height Do not support Purification protocol error Contact technical service. Purification protocol error Contact technical service. Wrong settings in the PIO.PSS file.

binary axis calculati- Contact technical support. on mode

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014

ERR_INVALID_MODULE_NO

Invalid Module No.

Missing file: define.pss deleted protocol error: a module number that is not defined in config.pss was assigned. Contact technical support

015

ERR_INVALID_ITEM_NO

Invalid Item No.

Missing file: define.pss deleted protocol error: an item number that is not defined in config.pss was assigned Contact technical support

016

ERR_NO_TIP

Cannot find tip

There are no tips in the Reaction Tip Tray Rack. The SW searches for tips three times before it indicates an error Check placement and number or reaction tips

017

ERR_INVALID_PARAMETER

Invalid parameter

purification protocol error Contact technical service.

018

ERR_INVALID_USER_PARAM

Invalid user parameter

purification protocol error: an expired general.gen file or an expired purification protocol is used Contact technical support

019

ERR_INVALID_IN_PORT

Cannot use this in-port Address

Wrong settings in the pio.pss file or wrong pio.pss file. Contact technical support.

020

ERR_INVALID_OUT_PORT

Cannot use this out- Wrong settings in the pio.pss file or wrong pio.pss file. port Address Contact technical support.

021

ERR_USER_BREAK

Forcibly closed by user.

the software was exited by the Windows NT task manager

The interlock error was cancelled by using CANCEL. All processes are suspended.

Close Results Screen and go back to Main Menu Screen. Click the Home Button. Then, reset all disposables/reagents and restart the run. Contact technical support 022 E ERR_Z_COMB_WARNING When Z is moved down, Nozzle may hit Comb 023 E ERR_INVALID_STR_VAL Invalid letters Hardware error: Drop catcher not moved back purification protocol error Call a service engineer. Purification protocol error Contact technical service. 024 E ERR_PSTACK_OVER Parameter Stack Over flowed 025 W ERR_2ND_INSTANCE Ignored 2
nd

Purification protocol error or internal software error Contact technical service.

BIOS

Software was started twice by the user MagNA Pure LC software was closed because of an error and the tMAn.exe process still exists: close it by WindowsNT task manager

execution

026

ERR_INVALID_KIND

Invalid Kind No

Purification protocol error: a module object that is not defined in Define.PSS file was assigned Contact technical support

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027

ERR_MINUS_VOLUME

Cannot assign minus volume

Purification protocol error Contact technical service. Reaction tip loss: check D-rings at nozzle head There are not sufficient tips in the Reaction Tip Tray Rack. The software searches for tips 3 times before it indicates an error. If this error is cancelled error 028 occurs. Check placement and number of reaction tips check O-rings at nozzle head

028

ERR_LOSE_TIP

Tip did not connect

029 030 031

E E E

ERR_NOT_BLOCK_SEQ ERR_INVALID_VOLUME ERR_INVALID_VOL_TABLE

No block sequence Invalid Volume Set Invalid Volume Table

Not applicable for MagNA Pure LC Instrument Not applicable for MagNA Pure LC Instrument purification protocol error: wrong general.gen file contact technical support Purification protocol error Contact technical service. Template error: wrong define.pss file contact technical support

032

ERR_1000P_OVER

Cannot assign more than 1000 pulse

033

ERR_NDEF_NAME

name=Not Defined

034

ERR_INVALID_PORT

Invalid Port No

Purification protocol error Contact technical service.

035 036

W E

ERR_USERSTOP_MOTOR ERR_VOLUME_OVER

User Stopped Motor Purification run was stopped by the user Liq in volume overflow Purification protocol error Contact technical service.

037

ERR_ARCNET_NORESPONSE

Interface is not responding. Time out.

Communication cable is disconnected. T-Net PC board is broken or hung-up

Check the connection between PC and instrument. When hung-up, turn off PC and MagNA Pure LC Instrument and restart both. If the error still appears, call a service engineer.

038

ERR_MOTOR_ALARM

Motor stopped by ALM or ERC signal

Hardware problem: the motor was stopped abruptly by the input of an alarm signal to the motor controller The main power has to be turned off or the power supply to motors has to be shut down by opening the door and restart of the instrument. If the error still appears, call a service engineer.

039

ERR_DROPCATCHER

Dropcatcher uncontrol

Hardware problem: the Liquid Drop Catcher did not move to the clot sensor 'on' position Call a service engineer to check the assembly of magnetic plate and make it move smoothly.

040

ERR_TIP_EJECT

Tip Eject Error

Hardware problem: when Tip_OFF command is to be executed, the Tip Eject plate did not move to the position of check sensor for Tip Eject Call a service engineer

042

ERR_NO_ARCNET

Script Stack Over Flow

Purification protocol error Contact technical service.

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043

ERR_STACK_OVERFLOW

Divided Zero. In Script Stack Command

Purification protocol error Contact technical service. Purification protocol error: an expired general.gen file or an expired purification protocol is used Contact technical support

044

ERR_ZERO_DIV

Not defined General Label

045

ERR_DOTDEF_GENERAL

Invalid Stack Command

Purification protocol error Contact technical service. Purification protocol error; invalid files Contact technical service.

046

ERR_INVALID_STACK

Invalid motor name

047

ERR_INVALID_MOTOT_NAM E

Invalid Tool No

Purification protocol error: invalid general.pss file Contact technical service.

048

ERR_INVALID_TOOL

Invalid VolumePulse Table

Purification protocol error: invalid general.pss file Contact technical service. Hardware problem: Peltier element of Heating Unit may be broken. Call a service engineer Hardware problem: Peltier element of cooling unit 1 may be broken. Call a service engineer Hardware problem: Peltier element of cooling unit 2 may be broken. Call a service engineer During the run, the fan stopped for more than 10 minutes. Call a service engineer to check whether the fan is broken. Reaction tip loss: check D-rings at nozzle head Check placement of reaction tips at nozzle head. Check O-rings at nozzle head.

049

ERR_INVALID_VPTABLE

Heat Block temperature is out of range

050

ERR_HEATBLOCK_ALARM

Cool Block1 temperature is out of range

051

ERR_COOLBLOCK1_ALARM

Cool Block2 temperature is out of range

052

ERR_COOLBLOCK2_ALARM

Ventilator Fan stopped

053

ERR_VENTILATOR_ALARM

Tip cannot be ejected

054

ERR_TIP_UNEJECT

Liquid handling cannot be executed correctly due to clots

Check samples for clotting. Check light conditions (see Chapter A, section 2.3.4 for details). Wrong Lysis Buffer used. Not applicable for MagNA Pure LC Instrument. This error appears only during execution of the surface scan due to an obstacle present on the Reagent/Sample Stage. Remove any obstacle from the Reagent/Sample Stage. Make sure that accessories and disposable plastics are placed correctly.

055 056

E F

ERR_NOTIP_INFO ERR_SCAN_SURFACE

No Tip Information Obstacle on Stage Plate. Please remove and start again.

057

ERR_DISP_REAGENT

The reagent currently dispensed is skipped

During vertical movement of the nozzle head the Reaction Tips hit an obstacle. Check that there is no obstacle like a Tub Lid Seal present on a Reagent Tub.

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175

2.

Maintenance
The following actions are performed during preventive maintenance:
1. Checks/Adjustments:

2.1 Preventive Maintenance It is recommended to perform preventive maintenance of the instrument the first time one year after installation. Subsequently, instruments should be maintained periodically with a one year interval.

Preventive Maintenance is performed by Roche Diagnostics service engineers only. MagNA Pure LC software 3.0 allows to set a so called 'Maintenance Reminder' by the service engineer. Several reminders for different maintenance tasks can be set to appear at a certain date or after a certain number of runs (e.g., for exchange of the nozzle D-rings).

Easy Teaching I/II/II Clot sensor signals Temperature settings (Heating Unit, Cooling Unit I/II) Leakage test Tip alignement Belt tension D-ring O-Ring Hepa filter UV-Lamp Air filter Clot sensor LM Guide Z-axis thread bar/slide shafts Z-plunger

2. Exchanges:

3. Cleaning/greasing:

If a service reminder appears on the screen call your Roche Diagnostics representative or service engineer.

2.2

Maintenance by the User

2.2.1 General Maintenance Measures The MagNA Pure LC system was designed to ensure safe operation. However, since human sample material may be processed in the instrument, a possible risk of infection exists. Therefore, when handling potentially infectious material, always follow standard safety procedures and take the following steps to minimize the chance of contamination or infection:

Note: After using bleach, ventilate the instrument for at

least 1 hour. After cleaning the Reagent/Sample Stage with bleach and water/ethanol, sterilize the instrument with UV light, as follows (see Chapter B, section 9.2 for details).

Always wear gloves and a mask when handling specimens and reagents. Keep the Waste Tip Slide clean. Never operate the instrument without a MagNA Pure LC Waste Bag secured to the end of the slide. Discard the Waste Bag and liquid waste bottle after preparing each full set of samples (32 samples). After sample preparation is complete: Remove and autoclave all disposables and other waste. Wipe the Reagent/Sample Stage with bleach, then with 70% ethanol and water (see Chapter A, section 9.2 for details).

Close the door and click the Decontamination button. Set the decontamination time (recommended setting: at least, 8 hours). Click the Start button.

Caution:

To avoid collisions between the robotic arm and the Cooling Blocks, ensure the blocks are seated correctly on the Reagent Stage. Press the Cooling Blocks firmly to seat the pins on the blocks in the holes of the Cooling Block 2 area! After each run, make sure the holes are clean. If necessary, clean these holes with a cotton swab!

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2.2.2 D-Ring Exchange Background The Reaction Tips are fixed to the nozzles of the nozzle head by elastic force using D-rings. Further purpose of the D-rings is to seal the inner compartment of the Reaction Tips airtight to avoide leakage and to ensure precise pipetting. Description of the D-Ring Exchange Tool

Pin inserted into the nozzle hole

D-ring pusher

After a certain period of usage D-rings might get worn out, causing leakage of liquid from the Reaction Tips or imprecise positioning of Reaction Tips at the nozzle leading to pipetting errors. If there are indications, possibly after performance of the Leakage Test (see following section for details), that D-rings are damaged or are loosing their stretch they should be exchanged against new ones. For D-Ring maintenance two 'D-Ring Exchange Kits' are available:

The D-ring Exchange Tool can be closed and opened like forceps:

D-Ring Exchange Starter Kit (Cat No 3 273 997), includes a booklet on how to change the D-Rings, the D-Ring Exchange Tool, 8 D-Rings, and grease. D-Ring Exchange Maintenance Kit (Cat No 3 274 004), includes a booklet on how to change the D-Rings, D-Rings (10 x 8 D-Rings), and grease.

Execution of the D-Ring Exchange

Start the MagNA Pure LC software as Standard User and click on the button 'Change D-Ring'. The robotic arm moves to a position above the front processing cartridge positions. You are now able to perform the D-Ring exchange by the use of the D-Ring Exchange Tool. Please follow the guidelines given in the following sections.

Appendix

177

Removing D-ring from the nozzle


Introduce the pin of the D-ring Exchange Tool into With your thumb pull down the part of the D-ring

the hole of a nozzle:

already pushed out:

Insert the pin as deep as possible:

By releasing the forefinger, detach the D-ring pusher

from the nozzle groove:

By keeping the position of the pin, move the D-ring

pusher sideward until it is inserted into the nozzle groove:

Remove the D-ring from the nozzle by holding it with

your thumb and pulling the D-ring changer downward:

Move the D-ring pusher further sideward with your

forefinger until the D-ring is pushed out of its groove:


The D-ring is now removed from the nozzle:

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Placement of new D-rings

To ensure correct reaction tip uptake and alignment it is essential to lubricate the new D-rings before placing them on the pipet nozzles. Furthermore, lubricating the D-rings prevents them from deterioration, such as cracking. The use of a special High Vacuum Grease is recommended. Such a grease is included in the two D-Ring Exchange Kits available.
Procedure for correctly lubricating the D-ring:

Note: It is advisable to check D-ring lubrication periodical-

ly (depending on the degree of utilisation at least once per month up to once per week), and add small amounts of vacuum grease as needed. Excess vacuum grease should always be removed with a kimwipe. It is recommended to change the D-rings at least after every 50 purification runs. The service engineer can set this time intervall in the 'maintenance reminder' function. You will then automatically be reminded to change the D-rings.

1. Apply a small bit of vacuum grease to index finger, rub between thumb and index finger. 2. Rub new D-ring between thumb and index finger. A slight amount of excess vacuum grease on the D-ring is acceptable during installation of new D-rings. 3. Install D-ring on pipet nozzle using D-ring changing tool and following the instructions below. 4. After D-ring is installed, wipe off excess vacuum grease with a kimwipe. After installation of new D-rings with correct lubrication, pipet tip configuration on nozzles should be correct, and Z-crashes should no longer occur. In summary, lubrication of D-rings is required for correct function including: correct pickup and ejection of Reaction Tips by robotic pipettor nozzles prevention of D-ring deterioration, such as cracking maintenance of tight seal to insure consistent and accurate pipetting
Set a new D-ring on the shaft of the D-ring Exchange

It also very important that the nozzle head with D-rings attached is not cleaned using bleach or ethanol. These reagents will remove grease from the D-Rings and most probably even from within the D-ring material which in turn will lead to enhanced detoriation of the D-rings. We completely advise against cleaning the nozzle head with these reagents. At least, all D-rings should be removed before cleaning. If after changing and lubricating the D-rings Z-crashes still occur this might be a sign that the O-Rings of the syringe plungers are detoriated. In this case call a Roche Diagnostics service engineer.

By using your thumb and forefinger, hold a D-ring

Tool:

and move to up along the shaft. Keep moving up until the D-ring is inserted to the nozzle groove:

Insert the pin of the D-ring Exchange Tool as deep as

possible into the nozzle hole:

Appendix

179

2.2.3 Leakage Test Purpose The Leakage Test is performed to detect possible leakage of air into the Reaction Tips, which in the worst case might lead to loss of liquid. Detection of leakage points towards a damage of the nozzle D-rings. Use It is recommended to perform the Leakage Test once per month.

The Start Information Screen appears as follows:

Although tear-and-wear of the D-rings depends on the number of isolations and Post Elution runs performed, it is also influenced by environmental conditions (light, chemicals etc.). Because of this, we recommend regular leakage testing, even if the instrument is not used regularly. If leakage is detected the nozzle D-rings should be exchanged. Also, it is recommended to perform the Leakage Test whenever the D-rings were exchanged, to verify their correct function.
Execution of the Leakage Test Start the MagNA Pure LC software as Standard User and open the Sample Ordering Screen

Put a Large Reagent Tub filled with 15 ml of water into Reagent Tub Rack position R1_L
Important: Make sure that the water has a temperature

of 20C 5C.

Put a Tip Tray of Large Reaction Tips into Reaction Tip position L1 (8 Large Reaction Tips are needed). Place a medium or small Reagent Tub covered with a Reagent Tub Lid Seal (without a Reagent Tub Lid) into position R8_S/M.
Note: This disposable is not indicated on the Start In-

Load the Sample Order File 'Leakage.sod'. The Sample Ordering Screen will appear as follows:

formation Screen. Place the Disposable Waste Bag and the Drop Catcher disposable. All other disposable plastics indicated on the Start Information Screen have not to be in place in order to perform the Leakage Test. Just confirm as if they were placed. Start the Leakage Test by clicking the 'OK' button.

It is not necessary to enter Sample and Elution Volume. Directly click the 'Start Batch' button to proceed to the Start Information Screen.

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Test procedure
Stage 1 Description After the start of the batch run, the robotic arm picks up Reaction Tips and moves to Reagent Tub Rack position R1_L to aspirate 1 ml of water plus an additional 100 l of air. 2 3 The robotic arm moves to a position over Reagent Tub Rack position R8_S/M. The nozzle head moves down to a position at which the water borderline within the Reaction Tips is in line with the upper rim of the Magnetic Plate behind the tips. 4 The nozzle head waits in this position for 10 min. It is now possible to open the instrument door, which eases observation of the nozzle head. 5 To recognize possible leakage watch the waterline inside the tips. A waterline constant over all tips will tell you that no leakage occurs. If you observe different waterlines from tip to tip or drops of liquid on the Reagent Tub Lid Seal in position R8 the nozzle D-rings should be exchanged. Note: A difference between the waterlines of up to 4mm can be tolerated and does not demand exchange of the D-rings . 6 After 10 min you are requested by the software to close the instrument door again. The water is then dispensed back into the Large Reagent Tub and the Reaction Tips are discarded. 8 Attention: If you choose 'Stop Batch' during execution of the Leakage Test you have to discard the reaction tips afterwards: Click on the 'Tip Discard' function button on the Main Menu Screen. Otherwise the water contained in the reaction tips would be spilled inside the instrument during the initialisation process proceeding a following run.

Appendix

181

3.

Trouble Shooting Guide

Problem Purification run not possible to start the purification

Possible Cause

Recommendation

sample input in the Sample Cartridge Grato right, i.e. from position H to position A): if less than 8 sample are to be processed, sample no. 1 is then missing

follow the instruction about sample input given in Chapter B, section 4.2.6.

run from the Sample Ordering Screen phic was done in wrong orientation (from left

drops of liquid on the reagent/sample stage run stopped due to head crash sensor activation (error code 010/013)

leakage of reaction tips due to damaged or worn nozzle D-rings

check D-rings and exchange, if necessary follow the instructions about placement of accessories and disposables

Reagent Tub Lid Seal left on Reagent Tub placed on Reagent/Sample Stage

check correct placement of accessories and disposables according to the Start Information Screen

wrong kind of Reagent Tub used Medium Reagent Tub M20 used for storage of Dilution Buffer instead of Medium Reagent Tub M30

Reagent Tub Rack not placed correctly Tip Stands not inserted correctly Reaction Tips taken from refill packages might be bent

check Reaction Tips do not autoclave Reaction Tips from refill packages check even alignment of Reaction Tips when attached to the nozzles check D-rings of nozzle head

Reaction Tips not attached correctly to the nozzles

run stopped due to error 039 pipetting of wrong liquid volumes

Liquid Drop Catcher disposable not inserted correctly into the Drop Catcher channel

follow the instructions given in Chapter B, section 2.4.

Reagent Tubs not closed with Tub Lids => liquid loss due to evaporation of buffers/reagents

follow the instructions given in Chapter B, section 4.2.5

MGPs: Not vortexed before filling into Reagent Tub; filled too early into Reagent Tub => evaporation of isopropanol

Reagents/buffers were stored too long in Reagent Tubs => liquid loss due to evaporation

Reaction Tips taken from bulk packages might be bent

check Reaction Tips do not autoclave Reaction Tips from bulk packages check O-rings of nozzle head follow the instructions about placement of accessories and disposables check correct placement of disposables according to the Start Information Screen

Reaction Tips not attached correctly to the nozzles

pipetting of air

wrong Reagent Tub used

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Problem Purification run purification status of samples is 'Fail' due to clot detection (error 054)

Possible Cause

Recommendation

sample was clotted

use fresh or frozen blood treated with anticoagulants; mix sample before use; make sure samples do not contain

air was pipetted

solid particles see entry above only use buffers included in the MagNA Pure LC reagent kits follow the instructions given in Chapter A, section 2.3.4 and Chapter A, section 8. check and exchange O-rings, if necessary reduce amount of cells or volume of sample (blood); do not use more sample material than recommended in the working instruction of the respective isolation kit

wrong Lysis Buffer used failure due to too bright ambient light or light reflections impairing the clot detection system

purification status of samples is 'Fail' due to tip loss (error 028) Magnetic Glass Particles in sample eluates

damaged or worn O-ring of the nozzles samples contained too high amount of cells

if it is unavoidable to use high cell numbers shear cell suspension/ lysates to reduce viscosity (follow the instructions given in the working instruction of the respective isolation kit)

lipaemic blood was used Magnetic Glass Particles were clumped

vigorously vortex MGPs before filling them into the Reagent Tub; do not let stand MGPs inside Reagent Tubs for a longer period of time, always fill MGPs as the last reagent

hardware failure: Wrong positioning of reaction tips

call service engineer

blue colored eluate

precipitate in Lysis Buffer

Do not store Lysis Buffer in the refrigerator or freezer. Do not use if precipitates formed. Warm buffer to room temperature or in a 37C waterbath until precipitates have dissolved.

red colored eluate

blood sample was partially clotted, not causing activation of clot detection sensor; clots carried over to the Storage Cartridge

use fresh or frozen blood treated with anticoagulants

Appendix

183

Problem Purification run no or poor yield of nucleic acids

Possible Cause

Recommendation

isolation buffers/reagents are nonfunctional

Do not store any isolation reagent/buffer in the refrigerator or freezer (unless recommended explicetly in the corresponding working instruction).

All buffers should not be used if precipitates had formed. Warm the buffers to room temperature or in a 37C waterbath until precipitates have dissolved.

contamination of reagents or disposables with nucleases (DNases, RNases)

follow general guidelines for contamination-free working do not wear powdered gloves; the powder might be contaminated with nucleases

use nuclease-removing reagents such as LTK-008 or RNase Zap (see page 78 for details)

RNA Isolation Kit I/II: Isopropanol contains water storage of samples was not optimal

do not store isopropanol in Reagent Tub use fresh or frozen samples; avoid use of samples that were stored at room temperature for a longer preriod of time; blood cells sedimented due to too long storage time

a too high sample amount (cell number) was used:

check the sample amount stick to the recommended upper limit of sample amount (see working instruction of the respective MagNA Pure LC reagent kit for details)

A too high cell number might cause a too viscous sample lysate. High viscosity of the lysate probably causes bead clumping. Not all of the beads will be captured by the magnet and will then be carried over to the eluate. Clumping of beads also causes a loss of yield, because then not every bead is able to bind nucleic acid. Furthermore, due to a too high cell number a too great amount of proteins and cellular debris is released. The amount of Proteinase K is optimized for the recommended cell number. Proteins and other cellular substances might impair binding of DNA to the magnetic beads because it has to compete for binding sites with these other compounds. The result will be a loss of yield, too. Unsufficient lysis of the sample may lead to clogging of tips. In addition, the magnetic particles have a certain maximum binding capacity. The amount of particles per reaction is optimized for a certain volume of sample material and cannot increased due to instrument characteristics. Thus, increasing the cell number over the recommended limit will not lead to an increase in yield. If using blood samples not the used volume of sample is important but the number of cells within it. Depending on the status of the blood donor (e.g., inflammation, infection, leukemia, drug treatment) e.g., 200 l might contain more than 1 x 106 cells.

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Problem Purification run low OD 260/280 values (< 1.8)

Possible Cause

Recommendation

A ratio below 1.8 points towards a too high content of proteins impurities. This in turn points towards an incomplete Proteinase K digestion.

check the sample amount stick to the recommended upper limit of sample amount (see working instruction of the respective MagNA Pure LC reagent kit for details)

high OD 260/280 values (>2.0)

Carry over of particles (cellular debris, incomplete lysed sample material, magnetic particles) into the eluate causing turbidity

check the sample amount stick to the recommended upper limit of sample amount (see working instruction of the respective MagNA Pure LC reagent kit for details)

Post Elution 'Start Post Elution' function button is not accessible pipetting of wrong volumes Cooling Blocks too cold (stored in the freezer) causing wrong initial volumes

incorrect Windows user name

follow the instructions for Windows user name (see Chapter B, section 2.2 for details) do not store Cooling Blocks in the freezer for fast cooling, only keep them in the refrigerator

dirt has accumulated around the pilots of Cooling Unit 2 (large cooling unit) causing the Cooling Block not fitting properly anymore (too high)

after placing Cooling Blocks many times this may lead to abrasion of block material which accumulates at the pilots; meticulously clean the large cooling unit

air bubbles in wells of Storage Cartridge

always check Storage Cartridge for air bubbles and remove any bubble present manually

too high nucleic acid concentration of eluted samples; a too viscous sample might lead to pipetting errors

check nucleic acid concentration of eluted sample; dilute sample or use fewer sample material

volume of PCR reagents not sufficient wrong 96-well PCR plate wrong reaction tubes

check for correct volume of PostElution reagents follow the instructions given in Chapter B, section 7.3.1 only use 1.5 ml Sarstedt reaction tubes with screw cpas Check programming of Post Elution protocol Check installation of Cooling Block files call service engineer

Run-time error 91: Object variable with block variable not set

A Post Elution file was loaded which contains a Cooling Block not installed into the software

Cooling Block data was installed faultily into the software

the instrument specific data are damaged

Appendix

185

Problem PCR analysis no or poor amplification

Possible Cause

Recommendation

isolation buffers/reagents are nonfunctional, no nucleic acid was isolated PCR reagents stored for a too long time period in the Cooling Block during Post Elution

see entry under 'Purification Run' prepare and store PCR reagents shortly before start of Post Elution run

reagents (isolation reagents and/or PCR reagents) were not removed when decontaminating the instrument by the inbuild UV lamp false positive results contamination of instrument, accesories, or disposables with nucleic acids or sample material cross contamination air bubbles at the bottom of Sample Cartridge well

always completely remove all reagents from the Reagent/Sample Stage before activating the UV lamp check general guide lines for contamination-free working follow instructions for decontamination and disinfection during aspirating a volume from Sample Cartridge wells eventual air bubbles might burst causing formation of aerosols

Drop Catcher disposable not changed tip discard was deactivated during Post Elution when dispensing samples

follow instructions given in Chapter B, section 2.4.

4.

File Types Used in the MagNA Pure LC software


Content saved purification run result screen saved sample ordering parameters purification protocol file: Contains information on pipetting steps performed during the Prologue phase of a purification protocol purification protocol file: Contains information on pipetting steps performed during the batch run reagent kit file: Contains information on buffers/reagents of MagNA Pure LC nucleic acid isolation kits epilogue file: Contains information on pipetting steps performed during the Epilogue phase of a purification run Post Elution protocol file: Contains information on cooling blocks and cooling block positions used in a Post Elution run Post Elution protocol file: Contains information on pipetting steps performed during a Post Elution run (only software versions previous to 3.0)

File extension *.ird *.sod *.plg *.blk *.kit *.elg *.pep *.pes

*.per *.mod, *.mop

saved Post Elution result screen Cooling Block files

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5.

Ordering Guide

For latest information on MagNA Pure LC nucleic acid isolation kits, disposable plastics, accessories, and other related products, please visit us at : http://www.magnapure.com. To order, solve technical queries, find product information, or contact your local sales representative visit us at: http:// www.roche-applied-science.com. MagNA Pure LC System: Instrument, Accessories, Disposables, and Reagents:
Product MagNA Pure LC
1)

Cat. No Instrument 2 236 931

Pack Size 1 instrument plus accessories *)

*) MagNA Pure LC System Package contains: 1. MagNA Pure LC Instrument 2. Pentium PC with Windows 2000 Professional and MagNA Pure LC software, Version 3.0 3. 43 cm (17") Monitor 4. MagNA Pure LC Accessory Kit 5. MagNA Pure LC Cooling Block for LC Centrifuge Adapters 6. MagNA Pure LC Cooling Block for 96-well PCR Plate 7. MagNA Pure LC Disposables Starter Set for two complete fillings of the Reagent/Sample Stage 8. MagNA Pure LC Operator Manual
Product Accessories (separately available) MagNA Pure LC Cooling Block for LC Centrifuge Adapters MagNA Pure LC Cooling Block for LC Sample Carousel MagNA Pure LC Cooling Block for 96-well PCR Plate MagNA Pure LC Cooling Block for Reaction Tubes MagNA Pure LC Cooling Block for A-Rings Reagent Reservoir Rack Reagent Tip Rack (Large Tips) Reagent Tip Rack (Small Tips) Tip Waste Slide Liquid Waste Funnel Waste Bottle Tray Disposables MagNA Pure LC Reagent Tub (small) 3 004 066 MagNA Pure LC Medium Reagent Tub 20 MagNA Pure LC Medium Reagent Tub 30 MagNA Pure LC Reagent Tub (large) 3 004 040 120 tubs 3 045 501 50 tubs 3 004 058 150 tubs 150 tubs 3 253 767 3 253 775 3 253 783 3 253 791 3 253 805 3 253 813 1 rack 1 rack 1 rack 1 slide 1 funnel 1 tray 3 201 287 1 cooling block 2 189 666 1 1 cooling block 2 189 674 1 1 cooling block 2 189 704 1 2 190 664 1 1 cooling block with 32 LightCycler Centrifuge Adapters 1 cooling block Cat. No Pack Size

Appendix

187

Product MagNA Pure LC Tub Lid (small, medium) MagNA Pure LC Tub Lid (large) MagNA Pure LC Tub Lid Seal MagNA Pure LC Reaction Tip (large) MagNA Pure LC Reaction Tip (small) MagNA Pure LC Reaction Tip (large), Refill Package MagNA Pure LC Reaction Tip (small), Refill Package MagNA Pure LC Sample Cartridge MagNA Pure LC Cartridge Seal MagNA Pure LC Processing Cartridge MagNA Pure LC Tip Stand MagNA Pure LC Waste Bottle MagNA Pure LC Waste Bag

Cat. No 3 004 082

Pack Size 300 lids

3 004 074 3 004 104 3 004 171

120 lids 400 seals 960 tips (30 x 32)

3 004 180

960 tips (30 x 32)

3 004 228

1 000 tips

3 004 236

1 000 tips

3 004 112 3 118 827 3 004 147

120 cartridges 200 seals 160 cartridges

3 004 155 3 004 198 3 004 201

200 tip stands 100 bottles 200 bags 500 disposables

S.E.T.S. - Swab Extraction Tube System 3 315 568

MagNA Pure LC Reagent Kits 2) MagNA Pure LC DNA Isolation Kit I*) MagNA Pure LC DNA Isolation Kit I - Lysis/Binding Buffer - Refill MagNA Pure LC DNA Isolation Kit II (Tissue) MagNA Pure LC DNA Isolation Kit III (Bacteria, Fungi) MagNA Pure LC DNA Isolation Kit Large Volume 3 310 515 1 kit 96 isolations from 1 ml blood 192 isolations from 300500 l blood 288 isolations from 20200 l blood 192 isolations from blood cells 192 isolations from 5 x 106 culture cells MagNA Pure LC Total Nucleic Acid Isolation Kit 3 038 505 1 kit (192 isolations) 3 264 785 1 kit (192 isolations) 3 186 229 1 kit (192 isolations) 3 246 752 1 bottle with 70 ml 3 003 990 1 kit (192 isolations)

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Product MagNA Pure LC Total Nucleic Acid Isolation Kit Lysis/Binding Buffer Refill MagNA Pure LC Total Nucleic Acid Isolation Kit - Large Volume MagNA Pure LC RNA Isolation Kit I (Blood, Blood Cells)* MagNA Pure LC RNA Isolation Kit II (Culture Cells) * MagNA Pure LC mRNA Isolation Kit I*) MagNA Pure LC mRNA Isolation Kit I Lysis Buffer Refill MagNA Pure LC mRNA Isolation Kit II (Tissue) *) MagNA Pure LC mRNA HS Kit

Cat. No 3 246 779

Pack Size 1 bottle with 70 ml

3 264 793

1 kit (192 isolations)

3 004 007

1 kit (192 isolations)

3 018 997

1 kit (192 isolations)

3 004 015

1 kit (192 isolations)

3 246 744

1 bottle with 70 ml

3 172 627

1 kit (192 isolations)

3 267 393

1 kit (192 isolations)

1) MagNA Pure is a trademark of a member of the Roche group. 2) The purchase of these products does not convey any licenses or other rights for the performance of PCR.

Product

Cat. No

Pack Size

Additional Products for the LightCycler System and the MagNA Pure LC Instrument LightCycler Carousel Centrifuge (230 V) LightCycler Carousel Centrifuge (115 V) 3 030 512 1 centrifuge plus rotor 2 189 682 1 centrifuge plus rotor

Related Products Product Cat. No Pack Size

LightCycler 1) and Products for the LightCycler System LightCycler Instrument LightCycler Capillaries 2 011 468 1 909 339 1 instrument plus accessories 1 pack (8 boxes, each with 96 capillaries and stoppers) LightCycler Centrifuge Adaptors 1 909 312 1 set (32 adaptors in an aluminum cooling block) LightCycler Sample Carousel LightCycler Software 3 1 909 282 1 909 304 1 carousel 1 pack (containing CD, an Operators Manual and a mouse pad) LightCycler Relative Quantification Software LightCycler Probe Design Software 3 139 174 1 Software Package 3 158 527 1 Software Package

Appendix

189

Product LightCycler Kits for PCR 2) LightCycler DNA Master, SYBR Green I 3) LightCycler DNA Master, Hybridization Probes LightCycler FastStart DNA Master, SYBR Green I 3) 4) LightCycler FastStart DNA Master, Hybridization Probes
4)

Cat. No

Pack Size

2 015 099 2 158 817 2 015 102 2 158 825 3 003 230 2 239 264 3 003 248 2 239 272 2 158 833

1 kit (96 reactions) 1 kit (480 reactions) 1 kit (96 reactions) 1 kit (480 reactions) 1 kit (96 reactions) 1 kit (480 reactions) 1 kit (96 reactions) 1 kit (480 reactions) 1 kit (50 control reactions)

LightCycler Control Kit, DNA LightCycler Kits for RT-PCR 2) LightCycler RNA Amplification Kit, SYBR Green I 3) LightCycler RNA Amplification Kit, Hybridization Probes LightCycler Control Kit, RNA

2 015 137

1 kit (96 reactions)

2 015 145

1 kit (96 reactions)

2 158 841

1 kit (50 control reactions) 1 kit (96 reactions) 1 kit (96 reactions)

LightCycler RNA Master SYBR Green 3 064 760 LightCycler RNA Master Hybridization Probes LightCycler h-PBGD Housekeeping Gene Set LightCycler h-2M Housekeeping Gene Set LightCycler h-G6PDH Housekeeping Gene Set LightCycler h-HPRT Housekeeping Gene Set LightCycler h-ALAS Housekeeping Gene Set LightCycler h-Housekeeping Gene Selection Set 3 310 159 3 302 504 3 261 891 3 261 883 3 146 081 3 146 073 3 018 954

1 kit (96 reactions)

1 kit (96 reactions)

1 kit (96 reactions)

1 kit (96 reactions)

1 kit (96 reactions)

5 x 16 reactions

1) LightCycler is a trademark of a member of the Roche Group. The technology used for the LightCycler system is licensed from idaho technologies Inc, Salt Lake City, UT, USA. 2) Purchase of this product is accompanied by a limited license to use it in the Polymerase Chain Reaction (PCR) process for life science research in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee, i.e., an authorized thermal cycler. No rights for any application, including any in vitro diagnostic application, are conveyed expressly, by implication or by estoppel under patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd claiming homogeneous or real-time amplification and detection methods. 3) SYBR is a trademark of Molecular Probes Inc., Eugene, OR, USA 4) FastStart is a trademark of a member of the Roche group.

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Related Products Product Cat. No Pack Size

LightCycler Parameter Specific Kits 2) LightCycler Factor V Leiden Mutation Detection Kit LightCycler Prothrombin (G20210A) Mutation Detection Kit LightCycler Apo B Mutation Detection Kit (codon 3500) LightCycler ApoE Mutation Detection Kit (codon 112 and 158) LightCycler NAT2 Mutation Detection Kit LightCycler CYP29 Mutation Detection Kit LightCycler CK20 Quantification Kit LightCycler TP mRNA Quantification Kit LightCycler DPD mRNA Quantification Kit LightCycler TS mRNA Quantification Kit LightCycler HER2/neu DNA Quantification Kit LightCycler HER2/neu RNA Quantification Kit LightCycler TeloTAGGG hTERT Quantification Kit1) LightCycler TeloTAGGG hTR Quantification Kit1) LightCycler t(9;22) Quantification Kit LightCycler t(14;18) Quantification Kit LightCycler t(8;21) Quantification Kit LightCycler inv(16) Quantification Kit LightCycler Parvovirus B19 Quantification Kit
1)

2 212 161

1 kit ( 32 reactions)

2 236 842

1 kit ( 32 reactions)

3 004 708

1 kit ( 32 reactions)

3 004 716

1 kit (32 reactions)

3 113 914

1 kit (32 reactions)

3 266 982

1 kit (96 reactions)

3 118 835

1 kit (96 reactions)

3 136 965

1 kit (96 reactions)

3 139 957

1 kit (96 reactions)

3 137 104

1 kit (96 reactions)

3 113 922

1 kit (32 reactions)

3 051 200

1 kit (96 reactions)

3 012 344

96 reactions

3 012 352

96 reactions

2 207 206

96 reactions (approx. 30 tests)

3 062 651

1 kit (96 reactions)

3 051 218

1 kit (96 reactions)

3 051 226

1 kit (96 reactions)

3 246 809

1 kit (for a max. of 48 samples)

TeloTAGGG is a trademark of a member of the Roche group.

Appendix

191

Related Products Product Cat. No Pack Size

LightCycler Parameter Specific Kits2) LightCycler Hepatitis A Virus Quantification Kit LightCycler Bacillus anthracis Detection Kit LightCycler EBV Quantification Kit LightCycler HSV 1/2 Detection Kit LightCycler GMO Screening Kit LightCycler GMO Maize Quantification Kit LightCycler GMO Soya Quantification Kit Reagents for Labeling and dual-color Detection of Hybridization Probes LightCycler Red 640 NHS ester 2 015 161 1 vial for labeling =5 x 50 nmol oligonucleotides LightCycler Red 705 Phosphoramidite 2 157 594 1 vial for the synthesis of 10 oligonucleotides labeled at the 5-end (0.2 m scale) LightCycler Color Compensation Set 2 158 850 Set for 5 calibration runs 3 267 164 1 kit (128 reactions) 3 330 028 3 315 177 3 267 199 3 267 172 3 303 411 1 kit (32 reactions for a max. of 14 samples) 1 kit (48 samples) 1 kit (48 samples) 1 kit (128 reactions) 1 kit (128 reactions) 3 246 795 1 kit (for a max. of 48 samples)

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6.
A

Index
E
Ejector ................................................................................ 15 Elution Buffer .................................................................... 98 Elution Cartridge .......................................... 20, 47, 98, 106 Elution & Post-Elution Unit ............................................ 17 Error code ........................................................................ 118 Error Codes ..................................................................... 171 Error Log ......................................................................... 118 Error.log .......................................................................... 167

Accessories ............................................................. 21, 23, 81 Accessory Kit ..................................................................... 11 Ambient light .................................................................... 16 A-Ring Ordering ............................................................... 79 A-Ring Ordering Screen ................................... 23, 151, 154 A-Ring Ordering Workflow ........................................... 156

B
Barcode ........................................................................ 25, 27 Barcode Cartridge .............................90, 103, 150, 151, 154, 156 Cool Block ............................................... 121, 152, 158 Printer ...................................................................... 121 Reader ........................................................................ 94 Sample .............................................................. 151, 154 Batch Status Screen ................................................... 73, 115 BatchID ................................................................... 121, 157 Bleach .......................................................................... 82, 66

F
Front Door ........................................................................ 12

G
Gel electrophoresis ................................................ 53, 55, 56

H
Heating Unit ......................................... 17, 20, 32, 106, 123 Help Menu ...................................................................... 118 HEPA filter ............................................................ 13, 62, 66 Home position .................................................. 172, 80, 103

C
Capture Buffer .................................................................. 50 Chaotropic salt ............................................................ 46, 49 Clot Detection ................................................................... 15 Clot Detection System ...................................................... 15 COBAS Amplicor .............................................................. 23 Communication cable .................................................... 174 Cooling Unit 1 .........................17, 20, 32, 81, 106, 123, 146 Cooling Unit 2 .........................17, 20, 81, 86, 124, 128, 146 Cross contamination ........................................................ 60 Cultured Cells ....................................................... 52, 55, 57

I
Infectious sample material ......................................... 66, 68 Information Screen ................................................. 102, 151 Installation ......................................................................... 64 Isopropanol ....................................................................... 49

K
Kohrsolin ........................................................................... 85

L
LC Carousel Centrifuge .................................................... 25 Leakage Test .................................................................... 180 LightCycler capillaries ....................... 8, 9, 25, 134, 135, 148 LightCycler PCR ............................................................... 52 LightCycler RT-PCR ............................................. 54, 55, 57 LightCycler SAM-file .............................................. 148, 149 LightCycler Sample Carousel ........................................... 23 Liquid Drop Catcher ............... 13, 37, 81, 87, 103, 172, 174 Liquid Waste ...................................................................... 43 Bottle ............................................................ 20, 22, 113 Discard ............................................................... 93, 160 Discard Bottle ............................................................ 13 Funnel ...................................................... 20, 22, 81, 85 Funnel Tray ................................................................ 81 Liquid Waste Discard ....................................................... 80 Log-In .......................................................................... 75, 78 Lysis/Binding Buffer ............................................. 48, 49, 50

D
Decontamination .................................. 66, 79, 82, 161, 176 Dilution Volume ............................................................... 98 Dispo Lockbar ............................................. 20, 83, 104, 114 Disposable Plastics ................................................ 29, 31, 44 Disposable plastics .......................................... 104, 105, 114 Disposables ........................................................................ 11 Disposables Starter Set ...................................................... 11 DNA Isolation Kit I .......................................................... 52 DNA, Isolation of .................................................. 46, 48, 53 DNase ................................................................................ 50 Door, Lock ................................................................ 80, 103 D-Ring ....................................................................... 15, 180 D-Ring Exchange ............................................................ 177

Appendix

193

M
MagNA Pure LC Cooling Block ............................ 20, 23, 24 A-Ring .............................................................. 128, 150 for 96-well PCR Plate .................................................21 for LightCycler Centrifuge Adapters ........................21 Installation ............................................................... 163 MagNA Pure LC Cooling Blocks ........................81, 86, 127 Magnetic Glass Particles ........................................ 46, 48, 49 Magnetic particles ................................... 37, 47, 73, 84, 110 Magnetic Plate ...................................................................12 Main Switch ................................................................. 14, 74 Maintenance .................................................................... 176 Maintenance Reminder ................................................... 176 Messages ........................................................................... 162 mRNA Isolation Kit I ........................................................57 mRNA, Isolation of ...........................................................50

N
Nozzle ................................................................................15 Nozzle Head ................................................................. 12, 15 Nozzle head .............................................................. 172, 174 Nucleic Acid Isolation Kits ...............................................51

Protocol file ......................................................120, 140 Protocol, Edit ...................................................121, 141 Result file ..................................................................121 Result Screen ............................................................147 Save ...................................................................120, 140 Select Cooling Block ................................................127 Single-dispensing .....................................................132 Start ...........................................................................146 Tip discard ................................................................130 Post Elution Steps Screen .................................. 73, 118, 119 Processing Cartridge ............... 17, 20, 38, 47, 106, 115, 160 Processing Unit ..................................................................17 Program Start ....................................................................78 Prologue unit .....................................................................83 Prologue Unit ....................................................................17 Proteinase K ............................................................... 48, 110 Protocol Installer .............................................................165 Purification Protocol ....................... 44, 92, 95, 97, 115, 165 Purification run ..... 9, 73, 114, 115, 116, 117, 118, 158, 167 Purity ............................................................................52, 55

R
Reaction Tip .......................... 39, 40, 73, 108, 138, 173, 177 Large .........................................................................125 Rack ................................................................ 21 ,81, 84 Refill ........................................................... Package 108 Tray ...........................................................................108 Trays ...........................................................................83 Reaction Tips ................................................... 15, 17, 19, 37 Reaction tubes .....................................................................9 Reaction Tubes 1 ........................................................ 27, 134 Reagent Tub ........................................... 34, 37, 73, 104, 109 Lid ...............................................................................36 Lid Seal ............................................................... 87, 109 Rack ...................................... 19, 21, 34, 35, 81, 84, 109 Rack, Positioning Frames ........................................110 Reagent Tubs ...........................................................9, 19, 35 Reagent/Sample Stage ............ 13, 17, 18, 19, 31, 39, 40, 41, ........................................... 66, 73, 80, 81, 102, 104, 105, 172 Reproducibility .......................................... 52, 54, 55, 57, 59 Result Screen ...................................................... 91, 117, 119 Load ..........................................................................117 Print ..........................................................................117 Save ...........................................................................117 RNA Isolation Kit I ............................................................54 RNA Isolation Kit II ..........................................................55 RNA, Isolation of total ................................................49, 56 Robotic arm .......................................................................12 RT-PCR reactions ............................................................134

P
Pager ID .............................................................................77 Paging unit .........................................................................77 Parvo Virus B........................................................... 19 59, 61 PBMCs ......................................................................... 54, 57 PCR-Plate ......................................................................... 127 PCR plates ........................................................................ 8, 9 PCR-plates ....................................................................... 134 Performance .......................................................... 52, 57, 59 Plasma ................................................................................59 Post Elution ........................... 8, 9, 86, 91, 92, 119, 152, 158 Define Name/Set Volume ................................122, 129 Delete ........................................................................ 120 Dilution series .......................................................... 139 Discard Tips ..................................................... 124, 136 Dispensing Volume ......................................... 124, 133 Estimated Total Time .............................................. 125 Initial volume ................................................... 130, 136 Load .................................................................. 120, 140 Mixing .............................................................. 124, 138 Multi-dispensing ...................................... 135, 137, 142 New ................................................................... 120, 126 Print .......................................................................... 120 Programming ................................................... 124, 132 Programming, Pause ............................................... 134 Protocol Description Table ..................................... 122

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S
Sample Cartridge .............17, 19, 32, 73, 83, 94, 98, 99, 111 Sample Name .................................................................. 100 Sample name...................................................................... 99 Sample Order Edit ........................................................................... 100 Load .................................................................... 90, 100 New ............................................................................ 90 Print ................................................................... 90, 101 Save ..................................................................... 90, 101 Table ........................................................................... 94 Sample Ordering ............................................... 9, 73, 79, 89 Sample Ordering Screen ........................................... 89, 119 Sample volume ............................................................ 93, 98 Sample Volume, Lysed ............................................... 93, 98 Scalability ........................................................ 53, 54, 56, 58 Serum ................................................................................ 59 Specifications .................................................................... 62 Stage Layout Graphic .............................................. 102, 104 Standard User .............................................................. 75, 78 Standard Users Main Menu Screen ................................ 79 Start Information ................................................................ 9 Start Information Screen ............................ 73, 94, 105, 157 Status Indicators ............................................................. 104 Status indicators ................................................................ 81 Storage Cartridge ...............................9, 20, 47, 98, 106, 123 Streptavidin Magnetic Particles ............................... 50, 110 Streptavidin-coated magnetic particles ........................... 46 Surface scan ..................................................................... 114 System Package ................................................................. 10

T
Text messages .................................................................... 80 Tip Stand .................................................................... 41, 107 Tip Waste Bag ................................................................... 13 Tip Waste Disposal Bag .................................................... 20 Tip Waste Disposal Slide ...................................... 13, 22, 81 Total Nucleic Acid Isolation Kit ....................................... 59 Total RNA isolation .......................................................... 54 Tub Lid Seal ....................................................................... 37

U
User Administration Add New User ............................................................ 77 Delete a user ............................................................... 78 Delete Password ......................................................... 78 User Name ................................................................. 77 User Administration Screen ............................................. 77 User Administrator ........................................................... 75 User Administrators Main Menu Screen ....................... 76 UV lamp .................................................. 12, 66, 79, 82, 161

W
Wall Spacers ...................................................................... 13 Waste Bag .................................................................. 42, 112 Waste Bottle Tray .............................................................. 22 Waste Disposal Slide ......................................................... 85 WBCs ........................................................................... 54, 57 Whole Blood ................................................... 52, 54, 57, 59

Y
Yield ....................................................................... 52, 55, 57

Appendix

195

Roche Applied Science

MagNA Pure LC Operators Manual


Version 3 Addendum 1 February 2005

Information regarding MagNA Pure LC Software Update 3.0.11

Please read the following information, which updates information given in the MagNA Pure LC Instrument Operator's Manual !

Dear valued user of the MagNA Pure LC Instrument,


In January 2005, Roche Applied Science introduced an updated version of MagNA Pure LC Software: Version 3.0.11. This latest version fixes some bugs found in previous versions and provides improved control commands for the instrument's central pipetting processing unit. Thus, MagNA Pure LC Software Version 3.0.11 increases the consistency, reliability and accuracy of MagNA Pure LC pipetting operations. You will not notice any changes in the usability and functionality of the user interface, since these are unchanged from the previous version (software version 3.0). However, due to these software improvements, you may notice the following change in the way the instrument works: After the instrument pipettes the eluted nucleic acid samples from the Elution to the Storage Cartridge, a small volume may remain in the wells of the Elution Cartridge. This will not affect the way the eluted nucleic acid is used in analytical procedures. Also, to accommodate the new software, please make the following change in the way you maintain the instrument: Grease the nozzle O-rings at least after every 10th purification run or if the instrument has not been operated for a longer period of time! (This is a change from the instructions given in the original maintenance section.) The Liquid Dispensing Accuracy and the Elution Volume Accuracy (*as specified in the table below) can only be guaranteed if the MagNA Pure LC Instrument is installed properly and maintained according to the instructions given in the Operator's Manual (Chapter C, section 2, Maintenance) and in this supplement.

Typical Instrument Specifications


With the introduction of MagNA Pure LC Software Version 3.0.11, the instrument specifications (given in the Operator's Manual on page 62) have changed slightly. The new specifications are:
Instrument Type Sample Capacity Liquid Dispensing Capacity Liquid Dispensing Accuracy* Elution Volume Accuracy* User Interface Power Source Power Consumption Dimension Weight Benchtop stand-alone instrument Up to 32 tests per batch 5 to 1000 l 5 to 100 l: < 3% CV 100 to 1000 l: < 2% CV > 90% (mean value; CV 5%) of the elution volume defined in the purification protocol User-friendly interface, based on Microsoft Windows 2000 operating system 100 to 240 V AC ( 10%); 800 VA; 50 to 60 Hz 800 VA max 100 mm x 650 mm x 890 mm (W x D x H) 151 kg

MagNA Pure LC Operators Manual Version 3.0 - Addendum 1 February 2005

MagNA Pure LC Operators Manual


Version 3 Addendum 2 August 2006

www.roche-applied-science.com

Important Additional Information on the UV Decontamination Procedure

Carefully read the following information, in addition to that given in the MagNA Pure LC Instrument Operator's Manual!

Dear valued user of the MagNA Pure LC Instrument,


Decontamination of the MagNA Pure LC Instrument Reagent/Sample Stage area is recommended on a daily basis to minimize the chance of contamination or infection. The daily decontamination procedure includes cleaning the Reagent/Sample Stage with bleach and water/ethanol followed by a recommended minimum 8 hour UV decontamination cycle. The decontamination procedures recommended by Roche Diagnostics are described in detail on pages 66, 82, 161, and 176 of the Operator's Manual. Regarding the UV Decontamination procedure the following information is given on p. 161 of the Operator's Manual: "If you open the front door during active decontamination, the UV lamp is turned off automatically and the countdown is suspended until you close the door again."

Please note that this statement is not correct: If the instrument door is opened manually during the UV decontamination cycle, the UV light will turn off, but the remaining decontamination time will continue to count down. This aborted or interrupted UV decontamination cycle may not be detected by the user as there is no error message.

Due to this behaviour of the MagNA Pure LC Software, always perform the following actions to avoid incomplete decontamination which in turn may eventually lead to a sample contamination:
To ensure the appropriate time period is allowed for UV decontamination, do not open the door of the instrument

during an active UV decontamination cycle.


If the UV decontamination cycle needs to be interrupted for any reason, restart the UV decontamination to allow

for the appropriate UV cycle time.

If you have any further questions regarding this matter, please do not hesitate to contact our Technical Services Department at your best convenience. To call, write, fax, or email us, visit the Roche Applied Science home page, www.roche-applied-science.com, and select your home country. Country-specific contact information will be displayed.

0706-04926854001

MagNA Pure LC Operators Manual Version 3.0 - Addendum 2 August 2006

MagNA Pure LC Operators Manual


Version 3 Addendum 3 September 2007

www.roche-applied-science.com

Important Information about Instrument Specification and Precautions to prevent Contamination and Infection
Carefully read the following information, in addition to that given in the MagNA Pure LC Instrument Operator's Manual!

Chapter A:

2.

Description of the Instrument


Changes
The MagNA Pure LC Instrument may be used at voltage from 110 120 V and 220 240 V

2.3.3 Side View of the instrument - Description Left Side, page 14 Current version Fuses
The MagNA Pure LC Instrument may be used at voltages between 100 V and 240 V

7.
7.1

Specification
Typical Instrument Specifications, page 62 Changes
110-120 / 220-240 V AC (10%); 800 VA; 5060 Hz

Current version Power Source


100240 V AC (10%); 800 VA; 5060 Hz

8.
Power Source

Instrument Installation, page 64


Changes
Independent, grounded electrical power outlet which can supply 110-120 V AC, at 10 A and 50/60 Hz or 220-240 V AC, at 5 A and 50/60 Hz

Current version
Independent, grounded electrical power outlet which can supply 90 - 240 V AC, at 10 A and 50/60 Hz

9.
9.2

Warning and Cautions


Precautions to prevent Contamination and Infection, page 66 Changes
Wipe the Reagent/Sample Stage with a 10% (v/v) bleach solution (0.5% Sodium Hypochlorite) and let it react for 10 min. To remove the bleach completely, wipe the instrument surface with 70% ethanol, followed by pure water. Commercial liquid household bleach typically contains Sodium Hypochlorite at a concentration of 5.25%. A 1:10 dilution of typically household bleach will produce a 0.5% Sodium Hypochlorite solution.

Current version Concentration of Wipe the Reagent/Sample Stage with bleach (10% solution of sodiumhypochloride) and let it Bleach
react for 10 min. To remove the bleach completely, wipe the instrument surface with 70% ethanol, followed by pure water.

Chapter B:

2.

System Setup
Changes
For decontamination either 10% (v/v) bleach solution ((0.5% Sodium Hypochlorite) see detailed instructions in Chapter A, section 9.2) or the following commercially available reagents may be used (follow the working procedures given by the respective manufacturer):

2.3.1 Decontamination of the Workstation, page 82 Current version Concentration of For decontamination either bleach (10% solution of sodiumhypochloride; see detailed instructions Bleach
in Chapter A, section 9.2) or the following commercially available reagents may be used (follow the working procedures given by the respective manufacturer):

0907-05135290001

MagNA Pure LC Operators Manual Version 3.0 - Addendum 3 September 2007