- Materials
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- Q5 HS Master Mix (Q5)
- Midnight Primer Pool A (MP A)
- Midnight Primer Pool B (MP B)
- Consumables
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- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- 1.5 ml Eppendorf DNA LoBind tubes
- Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Cat # 0030129504) with PCR seals
- 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 Freed et al., 2020 using Primal Scheme. These primers are designed to generate 1200 bp amplicons that overlap by approximately 20 bp. These primer sequences can be found here.
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In the template-free pre-PCR hood, prepare the following master mixes in Eppendorf DNA LoBind tubes and mix thoroughly as follows:
Volume per sample:
Reagent Pool A Pool B Nuclease-free water 3.7 µl 3.7 µl Midnight Primer Pool A (MP A) 0.05 µl - Midnight Primer Pool B (MP B) - 0.05 µl Q5 HS Master Mix (Q5) 6.25 µl 6.25 µl Total 10 µl 10 µl For x24 samples:
Reagent Pool A Pool B Nuclease-free water 102 µl 102 µl Midnight Primer Pool A (MP A) 2 µl - Midnight Primer Pool B (MP B) - 2 µl Q5 HS Master Mix (Q5) 172 µl 172 µl Total 276 µl 276 µl For x48 samples:
Reagent Pool A Pool B Nuclease-free water 203 µl 203 µl Midnight Primer Pool A (MP A) 3 µl - Midnight Primer Pool B (MP B) - 3 µl Q5 HS Master Mix (Q5) 344 µl 344 µl Total 550 µl 550 µl For x96 samples:
Reagent Pool A Pool B Nuclease-free water 407 µl 407 µl Midnight Primer Pool A (MP A) 6 µl - Midnight Primer Pool B (MP B) - 6 µl Q5 HS Master Mix (Q5) 687 µl 687 µl Total 1,100 µl 1,100 µl -
Using a stepper pipette or a multichannel pipette, aliquot 10 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 in the PCR plate(s), taking care not to cross-contaminate different wells. 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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Mix by pipetting the contents of each well up and down.
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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
61°C
65°C15 sec
2 min
3 min
35Hold 4°C ∞ -
Optional actionIf necessary, the protocol can be paused at this point. The samples should be kept at 4°C and can be stored overnight.