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
- - - - -

stratagene site directed mutagenesis


  • Please log in to reply
3 replies to this topic

#1 SF_HK

SF_HK

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 193 posts
2
Neutral

Posted 05 May 2009 - 09:53 PM

Hi,

I've used this kit about twice to revert mutations and the success rate (mutation efficiency) is 100%. However, now i have to perform deletion of a nucleotide. Has anyonedone this, if so, how many cycles of the pcr reaction do you do? my vector= 5.6kb , insert size = 1000bp?

Furthermore, the kit suggests using the special NY+ broth. Has anyone used simple LB for transformation instead of NY+broth?

Thanks

#2 T C

T C

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 277 posts
0
Neutral

Posted 06 May 2009 - 12:47 AM

Hey,

Never used it for deletions but it should work. Since its deletion of one nucoleotide, just follow the same consitions as mutagenesis. Elongation for 6 min using 1min per kb.

I have used LB and it works.

Hope it helps.

Best,
TC

Hi,

I've used this kit about twice to revert mutations and the success rate (mutation efficiency) is 100%. However, now i have to perform deletion of a nucleotide. Has anyonedone this, if so, how many cycles of the pcr reaction do you do? my vector= 5.6kb , insert size = 1000bp?

Furthermore, the kit suggests using the special NY+ broth. Has anyone used simple LB for transformation instead of NY+broth?

Thanks



#3 SF_HK

SF_HK

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 193 posts
2
Neutral

Posted 06 May 2009 - 06:39 PM

Thanks.

You used LB instead of the NZY+ borth. Could you clarify what cmpetent cels did you use when you used LB. I was just wondering if teh competent cells provided by the kit, XL1-blue, only work well with NYZ+ broth or LB as well.

#4 T C

T C

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 277 posts
0
Neutral

Posted 06 May 2009 - 10:08 PM

Well........I used the XL-1 blue cells but the ones that I regularly use in the lab and not the ones which came with the kit. :rolleyes:

But It shouldn't make a difference if the competence is high. Actually when I started doing this some years back, I always got more colonies with my own comp cells as compared to the commercial ones so I don't use the commercial ones at all.

Hope it helps.

Best,
TC

Thanks.

You used LB instead of the NZY+ borth. Could you clarify what cmpetent cels did you use when you used LB. I was just wondering if teh competent cells provided by the kit, XL1-blue, only work well with NYZ+ broth or LB as well.






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

©1999-2013 Protocol Online, All rights reserved.