findley at u.washington.edu
Tue Aug 16 11:45:32 EST 1994
I am not sure what you are trying to do, but a paper came out several
years ago with this nice money-saving technique for cloning very large
If you want to make a longish oligo insertion, rather than making TWO
very large oligos, make ONE, but with one end SELF-PRIMING.
Allow the oligo to self-prime, synthesize the opposite strand with klenow
plus dNTPS, then cut the ends with restriction sites which you
incorporated (during oligo design) and clone the now ds fragment.
You will have to add a couple of extra bases at each end, beyond the
restriction recognition sites for good cutting, and make a small loop at
the self priming end (four T-residues is good). (resulting in a small
stem-loop structure at the self-priming end, which makes for better stem
formation than a simple hairpin.
Not my idea, but it works great!
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