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
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3 replies to this topic
#2
T C
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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
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
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
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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.
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
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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. 
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
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.
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.
