Spliced overlap extension help

maneesh maneesh at drunkenbastards.com
Thu Jan 17 20:08:29 EST 2002


Hi all,
I am currently trying to create a construct by reading through three
template fragments which have overlapping ends, i.e.

         (GFP)                        (terminator)
         1 (1.6kbp)                3(~250bp)
-------------------              --------
                ||||              |||
                 --------------------
                      2(1.5kbp)
                      (YFG)


Prime at the ends of 1 and 2, I get the overlap product(1+2), if I
prime at the ends of 2 and 3, I get the overlap product (2+3).
But I if I try to priming at the ends of 1 and 3 I do not get the
overlap product (1+3).

That is I can read through each overlap individually, but if I attempt
to read through both, I only get a high molecular weight smear on the
gel (something what it looks like when you have too much Mg2+)...

The overlap reigons are short (12 and 20bp), and the first is almost
entirely GC.  I'm using standard PCR conditions, and the individual
overlaps seem to amplify robustly under a range of template
concentrations, and thermal cycling conditions.

Has anyone out there tried reading through three fragments, and did
they run into similar problems and come up with solutions?  Any hints
on what sorts of things I should do (change my sequences?) would be
appreciated....




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