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

Plasmid gene mutation


  • Please log in to reply
1 reply to this topic

#1 manojupreti

manojupreti

    member

  • Members
  • Pip
  • 5 posts
0
Neutral

Posted 23 October 2004 - 07:56 AM

I have some Tn10 derivative trasposon in plasmid vehicle cloned in E.coli. Example of them are - Derivative 105,marker in transposon is CamR and venicle is plasmid pnK2284,and deirivative 108 ,Markers in transposon is CAmr ,Plac ,plasmid vehicle is pNk2887.
I want use them for random mutagenesis in Pseudomonas.The gene of my intrest is in Plasmid.
Can you please suggest the protocol for mutagenesis using these transposon to mutate plasmid gene.
Thanking you in advance

#2 tuckern

tuckern

    member

  • Active Members
  • Pip
  • 18 posts
0
Neutral

Posted 18 November 2004 - 04:18 AM

use very cheap taq to amplify the insert off a template plasmid. Taq is error prone and will introduce random mutations into your dna. design primers that have restricion sites in them (or add them yourself) so that you can easily whack your pcr products back into the vector.
Adding extra MgCl2 to your pcr increases the error rate (as does adding dmso).




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

©1999-2013 Protocol Online, All rights reserved.