nCoV-2019 McGill Artic PCR Protocol, V4.1 at 63C
Anne-Marie Roy, Shu-Huang Chen, Ioannis Ragoussis, Sarah J Reiling, Kayleigh Loranger
Abstract
This is the updated SARS-Cov-2 PCR Protocol, with the ARTIC V4.1 primers, that is currently being used at the McGill Genome Center.
Steps
Primer pool preparation
PRIMER POOL PREPARATION
If required resuspend lyophilised primers at a concentration of 100 µM each
The V4.1 pre-pooled primers in 1.5mL Eppendorf labelled tubes are labelled “Pool 1 (100µM)” or “Pool 2 (100µM)". The primers do not require additional preparation.
Multiplex PCR
MULTIPLEX PCR
In the extraction and sample addition cabinet add 5µL RT product to each tube and mix well by pipetting.
In the mastermix hood set up the multiplex PCR reactions as follows in 0.2mL 8-strip PCR tubes:
Component Pool 1 [10 uM primer] Pool 2 [10 uM]
Q5 Hot Start High-Fidelity 2X Master Mix 12.5µL 12.5µL
Primer Pool 1 or 2 (10µM pool 1+2) 3.7µL 3.7µL
Nuclease-free water 3.8µL 3.8µL
Total 20µL 20µL
Add 20ul of PCR mastermix to the 5 ul RT product of step 10.
Pulse centrifuge the tubes to collect the contents at the bottom of the tube.
Set-up the following program on the thermal cycler:
Step Temperature Time Cycles
Heat Activation 98°C 0h 0m 30s 1
Denaturation 98°C 0h 0m 15s 36
Annealing 63°C 0h 5m 0s 36
Hold 4°C Indefinite 1
PCR clean-up
PCR CLEANUP
Combine the entire contents of “Pool 1” and “Pool 2” PCR reactions for each biological sample into to a single 1.5mLEppendorf tube.
Clean-up the amplicons using the following protocol:
Add an equal volume (1:1) of AmpureXP beads to the sample tube and mix by pipetting.
Incubate for 5 min at room temperature.
Pellet on magnet for 5 min. Remove supernatant.
Add 200 ul of 80% ethanol to the pellet and wash twice.
Let the beads dry for 3 min.
Add 30 ul elution buffer and resuspend the beads. Incubate for 3 minutes.
Pellet on magnet for 5 min. Remove and keep eluate (30 ul).
Amplicon Quantification and normalisation
AMPLICON QUANTIFICATION AND NORMALIZATION
Quantify the amplicon pools using a fluorimetric dsDNA assay. (e.g: PicoGreen with a standard curve 0-200ng)
We expect following concentrations:
Pool 1+2 combined:
100-150 ng/ul for Ct 14-24
30-80 ng/ul for Ct 25-29
10-30 ng/ul for Ct 30-36
Nextera Flex Library Prep:
After quantification of Pool 1+2, take a new plate and add 150 ng of Pool 1+2 and add up with nuclease-free water to a total volume of 30 ul (= 5 ng/ul).
Nanopore Library Prep:
After quantification of Pool 1+2, take a new plate and add 200 ng of Pool 1+2 and add up with nuclease-free water to a total volume of 20 ul (= 10 ng/ul).
