Primer synthesis problems
Austin P. So (Hae Jin)
who at what.where
Sat Dec 4 22:37:34 EST 2004
You'd have to say what the sequence is of the 5'-end of the primers,
which I am assuming contains your SfiI site. They could potentially form
*very* stable hairpins, and if the end is 5-'OH, then this can I suppose
lead to some really funky repairing by the host. But this is just
But I'll mention two things:
1. Why don't you cartridge (reverse phase) purify your oligos prior to
deblocking so that you get more of your full-length?
2. Why do you digest overnight with SfiI?
Bummer that this is still plaguing you thus far...
Paul Shinn wrote:
> I synthesize our oligos on a 96-well Polyplex. Most
> of the oligos are 43 and 56mers used for PCR/cloning. There
> is a SfiI restriction site at the end of each oligo that
> allows us to directionally clone our products into our home
> prepared vector using T4 DNA ligase. The vector/PCR junction
> sequence must be perfect and right now we are having some major
> issues with that.
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