TITLE: nCoV-2019 McGill Artic PCR Protocol,  V4.1 at 63C
AUTHORS: Sarah J Reiling, Kayleigh   Loranger, Anne-Marie  Roy, Shu-Huang  Chen, Ioannis  Ragoussis

[DESCRIPTION]

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]
SECTION: Primer pool preparation
1. PRIMER POOL PREPARATION

If required resuspend lyophilised primers at a concentration of 100 µM each
 

SECTION: Primer pool preparation
2. The V4.1 pre-pooled primers in 1.5 mL Eppendorf labelled tubes are labelled “Pool 1 (100µM)” or “Pool 2 (100µM)". The primers do not require additional preparation. 
 

SECTION: Multiplex PCR
3. MULTIPLEX PCR

In the extraction and sample addition cabinet add 5 µL RT product to each tube and mix well by pipetting.
 

SECTION: Multiplex PCR
4. 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             12.5 µL                                   12.5 µL         
Primer Pool 1 or 2 (10µM pool 1+2)                      3.7 µL                                    3.7 µL             
Nuclease-free water                                                 3.8 µL                                     3.8 µL             
Total                                                                            20 µL                                      20 µL                  

Add 20ul of PCR mastermix to the 5 ul RT product of step 10. 
 

SECTION: Multiplex PCR
5. Pulse centrifuge the tubes to collect the contents at the bottom of the tube.
SECTION: Multiplex PCR
6. Set-up the following program on the thermal cycler:  

Step                        Temperature     Time                    Cycles

Heat Activation     98 °C        30 s    1
Denaturation         98 °C        15 s     36 
Annealing              63 °C        5 min     36
Hold                       4 °C            Indefinite              1
 

SECTION: PCR clean-up
7. PCR CLEANUP

Combine the entire contents of “Pool 1” and “Pool 2” PCR reactions for each biological sample into to a single 1.5 mLEppendorf tube. 

SECTION: PCR clean-up
8. 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). 

 

SECTION: Amplicon Quantification and normalisation
9. 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

SECTION: Amplicon Quantification and normalisation
10. 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).