- Consumables
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- SARS-CoV-2 primers (lab-ready at 100 µM, IDT)
- Q5® Hot Start High-Fidelity 2X Master Mix (NEB, cat # M0494)
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat # 0030129504) with heat seals
- 5 ml Eppendorf DNA LoBind tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
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- Multichannel pipettes suitable for dispensing 0.5–10 μl, 2–20 μl and 20–200 μl, and tips
- P1000 pipette and tips
- P200 pipette and tips
- Thermal cycler
- Microfuge
- Centrifuge capable of taking 96-well plates
- Ice bucket with ice
- Optional equipment
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- PCR-Cooler (Eppendorf)
- PCR hood with UV steriliser (optional but recommended to reduce cross-contamination)
- Stepper pipette and tips
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Primer design
To generate tiled PCR amplicons from the SARS-CoV-2 viral cDNA, primers were designed by Josh Quick using Primal Scheme. These primers are designed to generate 400 bp amplicons that overlap by approximately 20 bp. These primer sequences can be found here. Where we show example data outputs in this protocol, the same parameters were used to design primers to the human coronavirus 229E to provide guideline statistics.
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In the pre-PCR hood, prepare the following master mixes in Eppendorf DNA LoBind tubes and mix thoroughly as follows:
Per sample:
Reagent Pool A Pool B RNase-free water 9.63 µl 9.63 µl Primer pool A (100 µM) 0.37 µl - Primer pool B (100 µM) - 0.37 µl Q5® Hot Start HF 2x Master Mix 12.5 µl 12.5 µl Total 22.5 µl 22.5 µl For x24 samples:
Reagent Pool A Pool B RNase-free water 270 µl 270 µl Primer pool A (100 µM) 10.4 µl - Primer pool B (100 µM) - 10.4 µl Q5® Hot Start HF 2x Master Mix 350 µl 350 µl Total 630.4 µl 630.4 µl For x48 samples:
Reagent Pool A Pool B RNase-free water 530 µl 530 µl Primer pool A (100 µM) 20.5 µl - Primer pool B (100 µM) - 20.5 µl Q5® Hot Start HF 2x Master Mix 690 µl 690 µl Total 1240.5 µl 1240.5 µl For x96 samples:
Reagent Pool A Pool B RNase-free water 1060 µl 1060 µl Primer pool A (100 µM) 41 µl - Primer pool B (100 µM) - 41 µl Q5® Hot Start HF 2x Master Mix 1375 µl 1375 µl Total 2476 µl 2476 µl -
Using a stepper pipette or a multichannel pipette, aliquot 22.5 µl of Pool A and Pool B into a clean 96-well plate(s) as follows:
Plate location X24 samples X48 samples X96 samples Columns Pool A: 1-3
Pool B: 4-6Pool A: 1-6
Pool B: 7-12Pool A: 1-12
Pool B: 1-12Note: For x96 samples, Pool A is a separate plate to Pool B.
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Using a multichannel pipette, transfer 2.5 µl of each RT reaction from the RT plate to the corresponding well for both Pool A and Pool B of the PCR plate(s). Mix by pipetting the contents of each well up and down.
There should be two PCR reactions per sample.
Example for X48 samples:
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Seal the plate(s) and spin down briefly.
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Incubate using the following program, with the heated lid set to 105°C:
Step Temperature Time Cycles Initial denaturation 98°C 30 sec 1 Denaturation
Annealing and extension98°C
65°C15 sec
5 min
25–35Hold 4°C ∞ Note: Cycle number should be varied for low or high viral load samples. Guidelines provided by Josh Quick suggest that 25 cycles should be used for Ct 18–21 up to a maximum of 35 cycles for Ct 35, however this has not been tested here.