Reverse Transcription with the ImProm-II Reverse Transcription System (Promega)
Principle
Detailed protocol
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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).
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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.
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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
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Add 15 µl of Reverse transcription reaction mix into each reaction tube.
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Anneal: Incubate at 25°C for 5 minutes.
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Extend: Incubate at 42°C for 1 hour.
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Inactivate Reverse transcriptase: incubate the reaction tube at 70°C for 15 minutes.
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Store in fridge for a few weeks or at –20°C for several months.