Activity No.

2
LABORATORY OPERATIONS: DESIGN AND EQUIPMENT
OF A MOLECULAR LABORATORY

Molecular Biology and Diagnostics Laboratory

2nd Semester; SY: 2020-2021
MLS 3rd Year Students

Instructors:
Jacus S. Nacis
Mary Jane H. Aum
Ryan Jay G. Mostoles

For Lorma Colleges, College of Medical Laboratory Science Students Use Only!
No part of this laboratory manual may be reproduced in any form or by any means without permission from the
Instructors.

NAME/S: CALUB, EDWARD PIOLO

VALDEZ, ZEN ANGELO
ESPADA, ANDREI
HUFANA, KLEIN ASHLEY
NONAN, JHENICA
SUBANG, ANGIE
GROUP NO. 3 DATE OF SUBMISSION: MARCH 22, 2021
INSTRUCTOR: MR. RYAN JAY MOSTOLES, RMT

GENERAL INSTRUCTIONS:
1. Read and understand the activity objectives, discussion, and procedures of this laboratory
manual.
2. Answer all the questions for research in the laboratory report sheet. Edit this file and send it back
to your Instructor.
3. This is group work. Submit your output as a group. The group leader will designate the work for
each member of the group. There should be an equal contribution. Cite your references.
4. Encode your answers and submit a soft copy to your Instructor.
5. Should you have questions, clarifications, and concerns, consult your Instructor through available
communication tools or gadgets.

OBJECTIVE:
At the end of the activity, the students should be able to:

1. State the strategies to prevent cross-contamination in the molecular laboratory

2. Determine the sources of PCR contamination
3. Determine the concerns in the design of molecular laboratory

1
 4. Design a molecular laboratory (asynchronous activity – groupwork – capstone project)

LABORATORY REPORT SHEET

QUESTIONS FOR RESEARCH:

1. What are the strategies to prevent cross-contamination in the molecular laboratory?

1. Laboratory Construction
- Contamination prevention starts with the construction or set-up of a PCR laboratory. At
a minimum, two areas should be designated for PCR testing: Pre- and Post-PCR. One
room or area should be designated specifically for Pre-PCR. Optimally, this room
should be further divided into two areas, PCR master mix preparation and sample
preparation/addition to master mix.
2. Environmental Control
- These two areas (Pre-PCR and Post-PCR) should have independent environmental
control and not use common ductwork for air conditioning. Moreover, both rooms
should be equipped with air-lock doors. Note: If physical barriers or separate rooms
cannot be established for Pre-PCR and Post-PCR work, all efforts should be taken to
set up the work areas as far apart as possible. Lab techs should treat these two work
areas as if they are in separate rooms. Care should be taken to wear different personal
protective equipment (PPE) in each area.
3. Unidirectional Workflow
- The workflow of a molecular lab should continue in one direction only, i.e. Pre-PCR >
Post-PCR. PCR master mix reagents and samples that may contain templates for PCR
should be prepared in the pre-PCR room only.
4. Dedicated Consumables and Equipment
- Another way that we can minimize contamination in a PCR laboratory is by using
consumables and equipment dedicated to each room. Each room and/or work area
should have its own centrifuge, vortexers, pipettors, gloves, coats, etc. For example,
never “borrow” a post-PCR pipettor for use in a pre-PCR room without thoroughly
decontaminating the pipettor first.
5. Use of Aerosol-Resistant Pipettes
- When pipetting samples, even the most seasoned technician can create aerosols
without the proper pipetting technique and pipette tips. Aerosols can lead to cross-
contamination from sample-to-sample. Aerosol-resistant pipette tips have a barrier,
which acts as a seal when exposed to potential liquid contaminants, trapping them
inside the barrier. This protects the pipettes from any liquid contaminants.
6. Pipetting Technique
- In any molecular assay, proper pipetting technique is critical to the performance and
quality of your results. Moreover, correct pipetting technique can minimize
contamination between samples that can lead to false positive results. Proper pipetting
technique ensures that the accurate volume is aspirated and dispensed and avoids
splashing when dispensing liquid.
7. Frequently Changing Gloves
- A lab technician should always wear fresh gloves when working in a PCR area.
Change gloves frequently, especially if you suspect they have become soiled with
solutions containing template DNA.

2
 8. Aseptic Cleaning Technique
- Proper aseptic cleaning should be carried out periodically before and after PCR work.
This applies to all work surfaces including bench tops, pipettors, fridge/freezer handles,
and any other touch points.
9. Wipe Tests
- Periodic wipe tests should be implemented as a standard procedure to proactively
monitor the laboratory environment for contamination before it becomes an issue. At a
minimum, wipe tests should be performed on a monthly basis. This frequency should
be increased if any contamination is suspected.
10. Positivity Rate Monitoring
- It is standard practice to include a positive control to assure proper performance of
extraction and amplification and functionality of the reagents. A no-template control
(NTC) is used to check for the absence of contamination in the reagents, consumables,
and environment. In addition to positive and negative controls, laboratories should
establish a standard procedure to monitor positivity rate.

2. What are the sources of PCR contamination?

PCR contamination is present but difficult to ascertain. If a significant contamination problem

appears in a PCR laboratory, it is needed to walk through the procedure of testing for
contamination and, if necessary, replace all reagents. The PCR parameters considered for
potential sources of contamination include amplification setup, amplification product handling,
and DNA aerosol and storage. Carryover contamination is determined by the following
methods in our laboratory.

Cross contamination between samples

A large number of target organisms in sample handling may lead to pre-amplification sample
cross contamination. The sources of contaminants between samples are diverse and can all
contribute to the contamination of the finished PCR product. These sources may include
reagents, disposable supplies, sample carryover, improper handling procedures,

Cross contamination between nucleic acids

Cross contamination between nucleic acids is a major problem in all PCR laboratories. Nucleic
acids from organisms or plasmid clones derived from organisms that have been previously
analyzed and that may be present in large numbers in the laboratory environment could be a
source of contamination. Contaminants can also be introduced by unrelated activities in
neighboring laboratories. These sources of contamination are problematic as they may lead to
pre-amplification cross contamination

PCR product carryover contamination

The most important source of contamination is from the repeated amplification of the same
target sequence, which leads to accumulation of amplification products in the laboratory
environment. Even minute amounts of carryover can lead to false-positive results. A typical
PCR theoretically generates as many as 108 copies of the target sequence. If uncontrolled,
amplification products will contaminate laboratory reagents, equipment, and ventilation
systems. Carryover of previously accumulated amplified DNA is considered the major source
of contamination.

3
 3. What are the concerns in the design of molecular laboratory?

LABORATORY DESIGN OF REAGENT PREPARATION ROOM

● This room should be kept as far away as possible from any room where PCR
amplicons are detected or transferred.
● If possible, this room should be separated into two spaces to separate work with
controls and primers from the master mixes.
● The flow of analysts, samples and reagents should always go in one direction, from the
pre-PCR areas (sample reception, PCR set-up and template addition) to the positive,
post-PCR areas (instrument room). Where analysts need to return to pre-PCR areas,
they may be required to wear additional protective clothing such as hairnets and masks
to prevent ‘shedding’ post-PCR genomic material. This is a common requirement in
forensic laboratories analysing biological material

LABORATORY DESIGN OF SPECIMEN/SAMPLE PREPARATION ROOM

● Ventilation filters, and decontamination procedures must be used to minimize risk of

cross-contamination between specimens.

LABORATORY DESIGN OF PCR

● This room should be divided into two work spaces (pre- and post-PCR), if possible, to
minimize contamination of thermal cyclers from aerosols created where reaction
tubes/microplate wells are opened following the PCR reaction for detection by gel
electrophoresis. Note that if PCR analysis is real-time PCR, reaction tubes need not be
opened except to validate or clarify.

Each area has separate sets of equipment and supplies

● Refrigerator/freezer (manual defrost)

● Pipettes, filtered tips, tubes, and racks
● Centrifuge, timers, vortex
● Lab coat (color-coded), disposable gloves, safety glasses, and other PPE
● Cleaning supplies
● Office supplies Ventilation system

Dead air box with UV light – serves as a clean bench area

Reagent Prep – Single entrance, reagents used for amplification should not be exposed to
other areas

Specimen Prep – Specimens should not be exposed to post-amplification work areas

Size of each area should consider space for equipment and bench space needed for
preparation

Two Areas Only

4
Area 1 – Reagent prep, specimen prep, and target loading – use of laminar-flow hoods
Area 2 – Amplification/product detection

Alternative to Spatial Separation

● Class II biological safety cabinet
● Dedicated areas for each work phase
● Unidirectional
● Automated specimen processing station/closed tube amplification and detection system

Other Laboratory Design Considerations

● Temperature and humidity requirements
● Exhaust ventilation
● Water quality Electric outlet
● Back-up power system
● Eye wash Ergonomic assessment
4. As a group work, design a molecular laboratory. This serves as your capstone project. (use
extra bond paper if needed)

5
6
REFERENCES/SOURCES:

1. 10 Ways to Minimize Contamination in a Molecular Laboratory. (2014, October 14).

Luminex Corporation. https://www.luminexcorp.com/blog/10-ways-minimize-

contamination-molecular-laboratory/

2. INABA, H. (1989). Polymerase chain reaction (PCR). Blood & Vessel, 20(4), 365–367.

https://doi.org/10.2491/jjsth1970.20.365

3. Buckingham, L. (2012) Molecular Diagnostics: Fundamentals, Methods, and Clinical

Applications

Lee, R. (2015). Molecular Laboratory Design, QA/QC Considerations. Texas Department

of State Health Services. Retrieved from:
https://www.aphl.org/programs/newborn_screening/Documents/2015_Molecular-
Workshop/Molecular-Laboratory-Design-QAQC-Considerations.pdf

http://www.austincc.edu/mlt/mdtech/MLAB_2337_2014.pdf
4.

7
INDIVIDUAL CONTRIBUTION:
Name Contribution
Leader: ESPADA, Andrei 4
Members: CALUB, Edward Piolo 2
VALDEZ, Zen Angelo 3
HUFANA, Klein Ashley 1
NONAN, Jhenica 3
SUBANG, Angelika