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.
Robert M. Horton
http://126.96.36.199 Have a :) day
"Scotty, try flushing the radioactive waste into the ventilation system!"
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