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
- - - - -

saturation mutagenesis library problem again


  • Please log in to reply
2 replies to this topic

#1 Biogareth

Biogareth

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 67 posts
0
Neutral

Posted 09 September 2011 - 02:23 AM

I tried to saturate another single amino acid position, but the result showed almost abundant with wide-type nucleotide again (in the attachment).
So I am wondering what can I do for this situation?
Thanks in advance!

Attached Thumbnails

  • QQͼδ.png


#2 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,502 posts
252
Excellent

Posted 09 September 2011 - 03:31 AM

I would say that is entirely your wild type, but perhaps the baseline is more convincing in other parts of the sequence. How are you doing the cloning and elimination of the template? Is this done with Stratagene Quikchange kits? Usual problems: too much initial template, failure of the DpnI digestion, failure of the PCR (leading to selecting only the wild type). Tell us a lot more about EXACTLY how you are doing this.

#3 Biogareth

Biogareth

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 67 posts
0
Neutral

Posted 09 September 2011 - 04:28 AM

I would say that is entirely your wild type, but perhaps the baseline is more convincing in other parts of the sequence. How are you doing the cloning and elimination of the template? Is this done with Stratagene Quikchange kits? Usual problems: too much initial template, failure of the DpnI digestion, failure of the PCR (leading to selecting only the wild type). Tell us a lot more about EXACTLY how you are doing this.



Hi Phage434,
You are right it looks wild type. The procedure I used actully the same like before. The procedure works well for several times. But it is not always successful.
The elimination of PCR products should be Okay, because I also check DpnI-treated mixture by SDS-page (in attachment, plasmid size 5.2kb, megaprimer size is 1.7 kb).




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

©1999-2013 Protocol Online, All rights reserved.