RT-PCR

From Genome@Yale

10uL reaction volumes work very well and replicate variance is same as 25uL.

Be economical; minimize reaction volume.

For 10uL or less, use the heat sealer to seal the plates. This will minimize evaporation during PCR.

Roche FastStart

10uL reaction volume

1. Add 3uL (2ng) cDNA to each well
2. Add 2uL 0.75uM primer mix (each 0.75uM)
3. Add 5uL 2X FastStart SYBR mastermix
4. Mix well by pipetting

5uL reaction volume

1. Add 1.5uL (1ng) cDNA to each well
2. Add 1uL 0.75uM primer mix (each 0.75uM)
3. Add 2.5uL 2X FastStart SYBR mastermix
4. Mix well by pipetting

Cycling Parameters

1. 10 minutes at 95C
2. 40-45 cycles
   1. 15 seconds at 95C
   2. 45 seconds at 60C
3. melt curve (optional)