Two-step RT-PCR for amplicon generation

Perform the following in a clean pre-PCR hood or in a pre-PCR designated area. The hood and all pipettes/tips entering the hood should be cleaned with 10% bleach and 70% ethanol prior to use.

Reverse Transcription

1. For each sample, mix the following in a PCR tube (preferably individually-hinged strip tubes):

• 7uL RNA
• 1 uL random hexamers (50 ng/uL)

1. Mix by inversion then spin down.
2. Heat to 70 degrees Celsius for 7 minutes, then place on ice to prevent secondary structure from re-forming.
3. Add the following to each tube:

• 10 uL ProtoScript II Reaction Mix
• 2 uL ProtoScript II Enzyme Mix

Note: you can also make the this as a mastermix, and pipette once per sample.

1. Place on the thermocycler and run the following program:

• 25 degrees Celsius for 5 minutes **note: this step can be swapped for a 5 minute hold at room temperature.
• 48 degrees Celsius for 15 minutes
• 80 degrees Celsius for 5 minutes
• Hold at 10 degrees Celsius

Amplification PCR

1. Make the following mastermix in a 1.5 mL tube. You will need both a Pool 1 mastermix and a Pool 2 mastermix.

• 8 uL Q5 Reaction Buffer
• 0.8 uL 10 mM dNTPs
• 0.4 uL Q5 DNA polymerase
• 24.4 uL Nuclease-free water
• 2.4 uL of primer pool (either pool 1 OR pool 2)

1. Label 2 0.2mL PCR tubes for each sample.
2. Pipette 36 uL of mastermix into the corresponding tubes.
3. Add 4 uL of cDNA into the corresponding tubes.
4. Mix by inversion and spin down.
5. Place on the thermocycler and run the following program:

• Step 1: 98 degrees Celsius for 30 seconds (initial denaturation)
• Step 2: perform 40 cycles of
  • 98 degrees Celsius for 15 seconds
  • 65 degrees Celsius for 5 minutes
• Step 3: Hold at 4 degrees Celsius.

**note: If generating amplicons for Miseq run rather than MinION run, you should do only 35 cycles of amplification, since you will have additional cycles of amplification during library prep.