Aug 24, 2020
nCoV-2019 McGill Artic PCR Protocol, 5 ul RT and V3 only + LA1 at 63C
Forked from a private protocol
- 1McGill University;
- 2University of Birmingham
- Coronavirus Method Development Community
- McGill Genome Centre
Protocol Citation: Sarah J Reiling, Josh Quick, Ioannis Ragoussis 2020. nCoV-2019 McGill Artic PCR Protocol, 5 ul RT and V3 only + LA1 at 63C. protocols.io https://dx.doi.org/10.17504/protocols.io.bjkrkkv6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 12, 2020
Last Modified: August 24, 2020
Protocol Integer ID: 40305
Abstract
V3 only primers can be found here:
https://github.com/sarahreiling/artic-ncov2019/blob/master/primer_schemes/nCoV-2019/V3/nCoV-2019_V3only.scheme.bed
Materials
MATERIALS
Q5 High-Fidelity 2X Master Mix - 500 rxnsNew England BiolabsCatalog #M0492L
nuclease-free water
Fresh 80% Ethanol
Quant-iT™ PicoGreen™ dsDNA Assay KitInvitrogen - Thermo FisherCatalog #P11496
AmpureXP beads Beckman CoulterCatalog #A63880
Primer pool preparation
Primer pool preparation
PRIMER POOL PREPARATION
If required resuspend lyophilised primers at a concentration of 100 µM each
Note
V3 only primers for this protocol were designed using Primal Scheme and generate overlapping 400 nt amplicons. Primer names and dilutions are listed in the table below.
Generate primer pool stocks by adding 5 µL of each primer pair to a 1.5 mL Eppendorf labelled either “Pool 1 (100µM)” or “Pool 2 (100µM)”. Total volume should be 490 µL for Pool 1 (100µM) and 490 µL for Pool 2 (100µM). These are your 100µM stocks of each primer pool.
Make another primer pool named "Pool LA1 (100 µM)" that contains 5 µl of primer pairs 5, 17, 23, 26, 66, 70, 74, 91, 97, and 10 ul of primer pair 64.
Note
Primers should be diluted and pooled in the mastermix cabinet which should be cleaned with decontamination wipes and UV sterilised before and after use.
Dilute this primer pool 1:10 in molecular grade water, to generate 10 µM primer stocks. It is recommend that multiple aliquots of each primer pool are made to in case of degradation or contamination.
LA1 primer pool will be diluted to 1 µM primer stock.
Note
Primers need to be used at a final concentration of 0.015µM per primer. In this case both pools have 98 primers in so the requirement is 3.65µL primer pools (10uM) per 25µL reaction. For other schemes, adjust the volume added appropriately.
Multiplex PCR
Multiplex PCR
MULTIPLEX PCR
In the extraction and sample addition cabinet add 5 µL RT product to each tube and mix well by pipetting.
Note
The extraction and sample addition cabinet should should be cleaned with decontamination wipes and UV sterilised before and after use.
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] Pool LA1 [1 uM]
Q5 Hot Start High-Fidelity 2X Master Mix 12.5 µL 12.5 µL 12.5 µL
Primer Pool 1 or 2 (10µM pool 1+2; 1µM LA1) 3.7 µL 3.7 µL 3.7 µL
Nuclease-free water 3.8 µL 3.8 µL 3.8 µL
Total 20 µL 20 µL 20 µL
Add 5 ul RT product as mentioned in step 10.
Note
A PCR mastermix for each pool should be made up in the mastermix cabinet and aliquoted into PCR strip tubes. Tubes should be wiped down when entering and leaving the mastermix cabinet.
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 00:00:30 1
Denaturation 98 °C 00:00:15 36
Annealing 63 °C 00:05:00 36
Hold 4 °C Indefinite 1
Note
Cycle number should be 25 for Ct 18-21 up to a maximum of 36 cycles for Ct 35
PCR clean-up
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 1.5 mL Eppendorf tube. Keep Pool LA1 separate from the combined Pool 1+2 until after the clean-up!!
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).
Note
Amplicon clean-up should be performed in the post-PCR cabinet which should should be cleaned with decontamination wipes and UV sterilised before and after use.
Amplicon Quantification and normalisation
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
Pool LA1:
1-10 ng/ul for all Ct
After quantification of Pool 1+2 and Pool LA1, mix them together in following ratio: 89.8% Pool 1+2 and 10.2% Pool LA1. For this, take a new plate and add 135 ng of Pool 1+2 and 15.3 ng of Pool LA1, and add up with nuclease-free water to a total volume of 30 ul (= 150 ng or 5 ng/ul).