Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Reverse transcription PCR


  • Please log in to reply
1 reply to this topic

#1 chn09

chn09

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 42 posts
1
Neutral

Posted 06 May 2010 - 01:11 AM

Hello everyone
Has any one used the Fermentas RevertAidô 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

#2 patty4150

patty4150

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 12 May 2010 - 03:45 PM

Hello everyone
Has any one used the Fermentas RevertAidô 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.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.