Re; pfu and pcr

[Qianfa Chen]

didier (d-fesque at nimr.mrc.ac.uk) wrote:
: >> 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

Just add 0.5 uL of Taq polymerase at the end of PCR cycles with pfu and 
incubate at 72 oC for 10 min.  The A overhang is added and ready for TA 
cloning (efficient than blunt end ligation).  

: 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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