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Ligation and Site Directed Mutagenesis ?


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#1 Bilbo Baggins

Bilbo Baggins

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Posted 18 January 2011 - 07:29 AM

Hi

I have been trying to introduce point mutants into a kinase, using the standard SDM procedure. I am using the Quickchange components (rather than kit), Pfu Turbo, Rxn buffer, DNTP, Quick Sol. My entire vector is 10 Kb in size, and so far I have not been able to get a single colony after transformation. I use 20 min as extension time, for 18 cycles. The primers have the mutation right in the center (fwd and rvrse).

So I decided to replace Pfu with Phusion, and its appropriate buffer. Though I am yet to know the result of this alternate method, a friend of mine suggested me to do a ligation also, after phusion PCR (before DPN digestion). This leaves me with a question ---- Y do I need to do a Ligation with phusion ? As far as I know, the quickchange protocol does not mention any ligation before or after dpn digestion. And Pfu and Phusion are almost similar, though Pfu has fast processivity. By all means, should'nt we be required to so a LIGATION after Pfu quickchange PCR reaction also, since TAQ always produces linear fragments.

Any ideas ? Should I simply do the transformation from my Phusion PCR product (after DPN dig) without the necessity of ligation ??

THnx :rolleyes:




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