Re; pfu and pcr

didier d-fesque at nimr.mrc.ac.uk
Wed May 7 06:14:02 EST 1997


>> Subject: pfu for cloning PCR?
>> 
>> Date: 30 Apr 1997 17:08:55 GMT
>> 
>> Hi all, does anybody know if pfu can be used for cloning PCR. I know
>> taq
>> is suitful for it. The reason I am using pfu is that I has tons of
>> pfu. It
>> has tried several times by many person in our lab in different
>> experiments and seemed no success. I dont know if someone has similar
>> problems as I have.
>> Thanks everybody, I like you.
>> 
>> C.Lu
>> C.Lu at cellbio.duke.edu



hi, the same arise in our lab because people does not have look to the
manufacturer instruction, using pfu taq require that all the parameters
of the pcr be changed, in general you should increase the dNTP, primers
concentration and modify the elongation time at least 2min per kb. in my
hand pfu work quite well from yeast genomic DNA or plasmid (up to 3kb at
present!), the yield is 10 time lower than with other (amplitaq cetus)
but the quality is fine, as there is no A overhang, the blunt end
cloning is without problem (if you do not forget to phosphorylate the
pcr product!)
in summary, after fighting a few week with our taq, I am totally
satisfied with pfu (I should mention I have no personal interest in
startagene company!)

didier



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