Reverse Transcription (RT):

1. Prepare the following components in a sterile environment:

2µL of RNA (0.5µg/µL)

1µL of 5X Hybridization (Hyb) Buffer

1µL of 1ng Downstream Primer

2. Combine the above components and mix gently.

3. Place the mixture in a PCR machine.

4. Perform the following temperature cycling:

Heat to 90°C and hold for a few seconds, then

Cool to 43°C over 30 minutes.

5. After cooling to 43°C, add:

19.5µL of 1.25X RT Mix (pre-warmed to 43°C and pre-mixed with 0.5µL of MMLV enzyme)

Mix gently.

6. Incubate in the PCR machine

43°C for 30 minutes, then Heat to 95°C for 5 minutes, followed by an immediate cooling to 4°C, Hold the mixture at 4°C

7. Store cDNAa at -20ºC