PCR-Ligation

[RYBICKI, ED]

> ecently there has been a lot of discussion re long PCRs.
> Well extending this a bit, has anyone any good tips
> on PCR ligation? IE joining two overlapping DNAs by PCR
> using primers at the extreme ends of each molecule?
> 
My student recently made a geminivirus C1/C2 ORF splice product by 
simply PCRing up the two bits to be spliced (separately) using 
Dynazyme pol (not a Taq, so hopefully no bases added on 
non-templated), then Klenowing to fill in in case fragments were not 
fully elongated, then PNKing to phosphorylate 5' ends (primers were 
not phosphorylated), ligating as for blunt-ended frags, then PCRing 
with the two end primers, and "tip-stabbing" (aka core sample PCR) 
the desired product and re-PCRing.  Worked pretty well - product 
about a kb or so.
  _________________________________________________________________
 | Ed Rybicki, PhD          | The percentage you're paying is too  |
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