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
- - - - -

what is the effectors of MSP?

PCR products

  • Please log in to reply
3 replies to this topic

#1 Sj Chen

Sj Chen

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 09 June 2013 - 11:53 PM

Hi,

I am a new person in DNA methylation. I have bought a bisulfite modification kit for test of DNA methylation. First I did a control test, that I treated a 400bp PCR fragment with several CG site, then i did the PCR using U and M primers. The results of MSP in two time MSP were different. I found PCR products in the U and M primers amplification at second time. what is the reason, it may be incomletely converion? anyone can give me a help? thanks,
Shanjun

#2 pcrman

pcrman

    Epigenetist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,165 posts
67
Excellent

Posted 10 June 2013 - 10:24 AM

Quite interesting--you bisulfite modified PCR product. Did you purify the PCR product before the treatment. The inconsistency in repeats may be resulted from non-specific amplification of the primers. MSP is prone to mispairing especially if the CpG C is not at the very 3' end of the primer. In MSP, annealing temperature and cycling number are important and need to be optimized.

#3 Sj Chen

Sj Chen

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 12 June 2013 - 11:06 PM

Thanks. yes, I purified the PCR products and the M and U primers were designed by methprimer (plus CpG island prediction) of The Li lab.

#4 pcrman

pcrman

    Epigenetist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,165 posts
67
Excellent

Posted 14 June 2013 - 01:29 PM

Then, you can try optimizing PCR conditions to get consistent results, BTW, what is purpose of bisulfite treating PCR product - as a positive control?





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

©1999-2013 Protocol Online, All rights reserved.