cDNA Synthesis and Amplicon PCR

This step uses the Influenza A/B Primer Pool (IPP) to amplify 8 genomic RNA segments from each RNA sample to produce the amplicons that will be tagmented.

This portion of the protocol is designed to use one primer pool that targets the whole genome of influenza A and B viruses using universal primers designed to anneal to the ends of each genomic RNA in RNA or cDNA form.

• FSM (First Strand Mix)
• RVT (Reverse Transcriptase)
• IPM (Illumina PCR Mix)
• IPP (Influenza A/B Primer Pool)
• Nuclease-free water
• 1.7 ml tube
• 96-well PCR plates (1)
• Microseal 'B' adhesive seal
1. Prepare the following consumables:
• FSM—Invert to mix. Keep on ice until use. If necessary, vortex to mix and eliminate all precipitation.
• IPM—Invert to mix. Keep on ice until use. If necessary, vortex to mix and eliminate all precipitation.
• IPP—Invert to mix. Keep on ice until use.
• RVT—Invert to mix. Keep on ice until use.
2. Save the following IMAP-Flu PCR program on the thermal cycler:
• Choose the preheat lid option at 105°C
• Set the reaction volume to 25 µl
• 42°C for 60 minutes
• 94°C for 2 minutes
• 5 cycles of:
• 94°C for 20 seconds
• 44°C for 30 seconds
• 68°C for 3 minutes
• 35 cycles of:
• 94°C for 20 seconds
• 58°C for 30 seconds
• 68°C for 3 minutes
• 68°C for 5 minutes
• Hold at 4°C
3. Label a new PCR plate RT-PCR.
1. In a 1.7 ml tube, combine the following reagents to prepare RT-PCR Master Mix. Multiply each volume by the number of samples.

Reagent overage is included to account for small pipetting errors.

Reagent

RT-PCR Master Mix (µl)

IPM

15

FSM

3.2

IPP

1.2

RVT

1.0

Nuclease-free water

3.6
2. Add 20 µl RT-PCR Master Mix to each well of the RT-PCR plate.
3. Add 5 µl RNA to each well of the RT-PCR plate.
4. Seal and shake at 1600 rpm for 1 minute.
5. Centrifuge at 1000 x g for 1 minute.
6. Place in the preprogrammed thermal cycler and run the IMAP-Flu PCR program.