5.

Multiplex tiling PCR amplification

IMPORTANT
Do not vortex the NEB Q5 Hot Start Hi-Fi 2X MasterMix as it is very viscous. Pipet mix then
spin down

5.1. Multiplex PCR Master Mixes Preparation In:

Reagents
Retrieve and thaw Q5 Hot Start Hi-Fi 2X MasterMix and ARTIC primer vials and place on cold/ice
Out:
In separate microcentrifuge tubes, prepare the ARTIC set A and for ARTIC set B reaction mixes PCR MMix to 5.2.

ARTIC Set A ARTIC Set B

Mastermix Component
Vol/Rxn Vol/Rxn

Q5 Hot Start Hi-Fi 2X MMix 12.5 µL 12.5 µL

Nuclease-free sddH2O 5.8 µL 5.8 µL

10 µM ARTIC v4.0 Set A 4.0 µL -

10 µM ARTIC v4.1 Set A 0.2 µL -

10 µM ARTIC v4.0 Set B - 4.0 µL

10 µM ARTIC v4.1 Set B - 0.2 µL

Subtotal 22.5 µL 22.5 µL

5.2. Master Mix Dispensing In: PCR MMix from 5.1.

Out: PCR Plate Mix to
In a 96-well plate, dispense the reaction mix sets in the following manner 5.3.
IMG A
Dispense 22.5 µL of ARTIC Set A Rxn Mix starting from well A1, column-wise
Dispense 22.5 µL of ARTIC Set B Rxn Mix starting from well A7, column-wise

5.3. cDNA Template Addition In:

PCR Plate Mix from
5.2.
REMINDER cDNA from 4.3.

Bring forward the NEC and NTC reactions to monitor cross-contamination events
Out:
PCR Plate Rxn to 5.4
Retrieve the cDNA 8-tube strips, thaw, spin down and place on a cold rack
IMG-B
Use a P20 8-channel pipettor to perform template addition:

Transfer 2.5 uL template from a cDNA strip tube to wells containing ARTIC Set A Rxn Mix

Transfer 2.5 uL template from a cDNA strip tube to wells containing ARTIC Set B Rxn Mix

cDNA 8-TubeStrip 01 02 03 04 05 06

ARTIC Set A Rxn

A1-H1 A2-H2 A3-H3 A4-H4 A5-H5 A6-H6
Plate Column

ARTIC Set B Rxn

A7-H7 A8-H8 A9-H9 A10-H10 A11-H11 A12-H12
Plate Column

Seal wells using 8-cap strips, pulse vortex, and spin down with a plate spinner

5.4. cDNA Multiplex Tiling PCR Amplification In:

PCR Plate Rxn from
Load samples the ABI 7500 Fast thermocycler or CFX96 located in the PCR room. 5.3.

Use the SC2_30TD.EDS or SC2_30TD.pcrl and confirm the profile as shown Out:
Set A/Set B Amplicons
to 6.2
Cycle Step Temp Time Cycle
 Initial denaturation 98°C 0:30 1

Denaturation 98°C 0:15

65°C 25
Annealing/Extension 5:00
Δ-0.1°C**

Denaturation 98°C 0:15

5
Annealing/Extension 62.5°C 5:00

Final hold 4°C Inf hold

**Touchdown PCR: Initial anneal/extend temp of 65°C; -0.1°C/cycle for 25 cycles

Samples can be stored at –20°C if they are not used immediately