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stratagene site directed mutagenesis - deletion of single base nucleotide (May/05/2009 )

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

-SF_HK-

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

SF_HK on May 6 2009, 12:23 PM said:

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

-T C-

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.

-SF_HK-

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

SF_HK on May 7 2009, 08:09 AM said:

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.

-T C-