Reverse Transcription with the ImProm-II Reverse Transcription System (Promega)

April 22, 2011 08:02, Last update: May 03, 2011 13:18

Principle

Reverse Transcription with the ImProm-II™ Reverse Transcription System (Promega)

Detailed protocol

  1. In a 0.2 mL tube, combine 1 µl of c-DNA primers (Random primer or oligo(dT)) with 1 4 µg of RNA solution (up to 4 µL).

  1. Place the RNA-primers mixture into a preheated 70°C heat block for 5 minutes. Then immediately chill in ice for at least 5 minutes.

  2. Prepare the reverse transcription reaction mix as follows :

For the experimental reaction (for each sample):

ImProm-II 5X Reaction Buffer 4 µL

MgCl2 4.8 µL

dNTP mix 1 µL

Rnasin Ribonuclease Inhibitor 0.5µL

ImProm-II Reverse Transcriptase 1 µL

Nuclease-free Water 3.7 µL

 

For the negative control (for each sample):

ImProm-II 5X Reaction Buffer 4 µL

MgCl2 4.8 µL

dNTP mix 1 µL

Rnasin Ribonuclease Inhibitor 0.5µL

Nuclease-free Water 4.7 µL

  1. Add 15 µl of Reverse transcription reaction mix into each reaction tube.

  1. Anneal: Incubate at 25°C for 5 minutes.

  1. Extend: Incubate at 42°C for 1 hour.

  1. Inactivate Reverse transcriptase: incubate the reaction tube at 70°C for 15 minutes.

  1. Store in fridge for a few weeks or at –20°C for several months.