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| 发布时间：2007-01-04|
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cDNA preparation:
4-5µL RNA
3µL oligo dT primer
12µL DEPC-treated water
Incubate at 70 degrees for 10 minutes, then put on ice.
Add
8µL 5X 1st strand buffer (from Invitrogen or Gibco)
4µL 0.1M DTT
5µL dNTPs (from Ex-Taq kit)
1µL RNase inhibitor if desired
Incubate at 48 degrees for 2 minutes, then add
2µL reverse transcriptase
Incubate at 48 degrees for 60 minutes
cDNA can be frozen for later use or used immediately for PCR. You should do a no reverse transcriptase control for each sample when preparing cDNA. This will allow you to know if there is DNA contamination in your RNA samples when you do PCR.
PCR conditions:
1-5µL of cDNA
5µL 10X Ex-Taq Buffer
4µL dNTPs
2µL each primer
49µL water
0.5µL Ex-Taq
Use PCR program appropriate to the primers.
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Gene-specific RT-PCR