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 
guessing...

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...

Austin

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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