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
- - - - -

never get bands by pfu related enzymes


  • Please log in to reply
2 replies to this topic

#1 ginger

ginger

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 10 July 2010 - 11:20 AM

i'm going to do a fusion pcr.there is a 1.6 kb to be fused with another 1.6 kb .i never get this 1.6 band with pfu while taq amplify it on my template very well.i have changed my primers three times and also my enzyme.pfu easily amplifies b2m gene on my template(3kb


)while this 1.6 kb high gc content region will fail in pcr.what is ur suggestion to overcome this nerve-racking problem?if taq adds a at the end what should be done to remove them?
thanks a lot :P

#2 snoopyx

snoopyx

    member

  • Active Members
  • Pip
  • 24 posts
0
Neutral

Posted 12 July 2010 - 02:36 AM

I also had the problem that some seq just couldn't be amplified with pfu. Don't know why, never found out. If you want you can try to mix taq & pfu, my bench neighbor had good results with that method.
What are your downstream applications for the pcr-product? You can insert restriction sites if your worried about additional a's.

#3 ginger

ginger

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 12 July 2010 - 04:00 AM

I also had the problem that some seq just couldn't be amplified with pfu. Don't know why, never found out. If you want you can try to mix taq & pfu, my bench neighbor had good results with that method.
What are your downstream applications for the pcr-product? You can insert restriction sites if your worried about additional a's.




hi
thanx for ur reply. i thought its only me that has this strange problem! :lol: i have designed an overlap of 23 nucleotide for my forward primer to be fused with another fragment.since no one in our lab has experience about this pcr i have no idea if i do experiment with taq (because it adds a) it is probable that my fragments do not make hybrids(i'm not sure) :D . if i wanted to mix pfu with taq what kind of pcr condition i should use?pfu buffer plus enzyme mix or taq buffer plus enzyme mix? one of my friends has recently purchased a proofreading version of taq (LA taq ) it is faster than pfu with a lower proofreading activity but it adds a at the ends (has anybody in your lab used this enzyme?)
thanx everybody for participating in this forum i 'm glad to hear your experiences about this kind of pcr and pfu enzyme




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

©1999-2013 Protocol Online, All rights reserved.