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
- - - - -

SYBR melting curve vs RT-PCR gel


  • Please log in to reply
1 reply to this topic

#1 konnicska

konnicska

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 04 November 2009 - 02:19 PM

Hello,

I was trying to validated a custom designed taqman probe -- I ran a SYBR green melting curve and independently ran a RT-PCR reaction using the same RNA then ran the product on a gel. The interesting thing is, I got only 1 peak in the SYBR melting curve, which usually means that there's only 1 PCR product, but I got 3 distinct bands on my gel! Other than my <100 bp expected PCR product, I also saw a 200 bp and a 600 bp band. Any idea what's going on here?

Thanks,
Konnicska

Edited by konnicska, 04 November 2009 - 02:20 PM.


#2 Trof

Trof

    Brain on a stick

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,198 posts
109
Excellent

Posted 05 November 2009 - 03:06 AM

Did you use the same PCR conditions for both?

Usually it's not needed to run a separate reaction to put on gel, you can analyse your samples on real-time cycler and then load the same reaction right onto gel. SYBR doesn't interfere with EtBr staining. That way you know for sure, that what you see on the gel and on the melting matches.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon





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

©1999-2013 Protocol Online, All rights reserved.