SOE-PCR woes!

Robert Horton horton at biosci.cbs.umn.edu
Tue Jan 23 14:07:39 EST 1996


Chanda Eva R (3erc at QLINK.QUEENSU.CA) wrote:
: I've tried several times to join 3 PCR fragments together by SOE, or 
: splicing by overlap extension (a.k.a. megapriming), but without success! 
: I followed the "standard" protocol (Horton et al,1993. Meth.Enzymol 217: 
: 270-279)i.e. used 1/4 of a gel-purified, Genecleaned, 100uL PCR reaction of 
: each fragment, 1uM of each of the "outer" primers, 30 cycles of 1 min 
: @92C, 2 min @60C, 3 min @72C. My fragments overlap by 60 and 33 bp, 
: respectively, which is more than the protocol recommends--could that be 
: causing problems? (e.g. by annealing too slowly/inefficiently compared to 
: shorter overlaps) I'd GREATLY appreciate any practical tips anyone could 
: give me!! 

I don't think the overlap is too long; we've used overlaps longer than
100bp with no problem. If you could send me the sequences of the
primers and templates, I'd be happy to look them over.


-Bob

---
Robert M. Horton
http://134.84.47.3                                       Have a :) day

"Scotty, try flushing the radioactive waste into the ventilation system!"



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