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
- - - - -

PCR amplification with new restriction sites troubleshooting


  • Please log in to reply
2 replies to this topic

#1 Suzanna

Suzanna

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 15 April 2013 - 12:03 PM

Hi all

I'm using Phusion polymerase to amplify a 4.2 kb target in a pcDNA3 vector. I once used the same template amplify the target successfully in GC buffer and 5% DMSO , annealing temperature 57.5. I'm using new primers to amplify the same template from the same vector and with nearly the same Tms and recognition sequences. But this time I can't amplify the target even using GC buffer and DMSO. The reaction products are high molecular weight nonspecific products that resemble the products i previously got in HF buffer without DMSO.

The Tms for these are 62.9 (50 % GC) and 63.6 (50 % GC). Previous primers that worked were 62.9 (55.5% GC) and 63.7 (55.5 %GC).

Is it worthwhile to use my previous PCR product as template for this new reaction in case there are sites within the vector that are getting amplified giving spurious products? I'm really quite desperate. Help!! :(

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,237 posts
336
Excellent

Posted 15 April 2013 - 12:35 PM

I take it you have done Mg2+ and temperature gradients to optimise these primers?

#3 Suzanna

Suzanna

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 16 April 2013 - 09:38 AM

temp grads yes but magnesium no . I didn't do a magnesium grad because teh product is already non-specific and didn't see how magnesium would help increase specificity




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

©1999-2013 Protocol Online, All rights reserved.