Reverse transcription PCR - (May/06/2010 )
Hello everyone
Has any one used the Fermentas RevertAid™ Reverse Transcriptase for cDNA synthesis.
The kit recommends hetaing the RNA and primer mix at 65 degrees for 5 min. Im using total RNA from bacteria. My questions are as follows
1 Is bacterial RNA stable at 65 degrees? . Ive seen that the DNase treatment step of RNA that says that RNA hydrolyses at 65 degrees in the absence of a chelating agents.
2. Im not getting results with my RT-PCR. The RNA is intact and of good purity.
3. For the synthesis of first strand cDNA from the RNA, is it enough to add the reverse primer only, or are both forward and reverse primers necessary?
pls help
-chn09-
chn09 on May 6 2010, 02:11 AM said:
Hello everyone
Has any one used the Fermentas RevertAid™ Reverse Transcriptase for cDNA synthesis.
The kit recommends hetaing the RNA and primer mix at 65 degrees for 5 min. Im using total RNA from bacteria. My questions are as follows
1 Is bacterial RNA stable at 65 degrees? . Ive seen that the DNase treatment step of RNA that says that RNA hydrolyses at 65 degrees in the absence of a chelating agents.
2. Im not getting results with my RT-PCR. The RNA is intact and of good purity.
3. For the synthesis of first strand cDNA from the RNA, is it enough to add the reverse primer only, or are both forward and reverse primers necessary?
pls help
Has any one used the Fermentas RevertAid™ Reverse Transcriptase for cDNA synthesis.
The kit recommends hetaing the RNA and primer mix at 65 degrees for 5 min. Im using total RNA from bacteria. My questions are as follows
1 Is bacterial RNA stable at 65 degrees? . Ive seen that the DNase treatment step of RNA that says that RNA hydrolyses at 65 degrees in the absence of a chelating agents.
2. Im not getting results with my RT-PCR. The RNA is intact and of good purity.
3. For the synthesis of first strand cDNA from the RNA, is it enough to add the reverse primer only, or are both forward and reverse primers necessary?
pls help
number three: You only need the complement to your RNA for the first strand.
number two - I've had variable results with different kits, Check the expiry date on your kit. Is your enzyme being treated nicely (ie no freeze thaws etc)?
number one - not sure, but I would think that if the heat step is a problem then you most likely have RNAses. If the kit calls for a heat step, I'd bet RNA can be stable at 65.
-patty4150-