PC at no.email.sorry
Wed Feb 18 22:30:25 EST 2004
another problem might of course be depurination of the oligos.
I guess you cant use HPLC or PAGE for each and every oligo you have make...
good luck anyway,
Paul Shinn wrote:
> P.C. <PC at no.email.sorry> wrote:
> : that happens only with this pair of primers?
> : my explanation - something was wrong with the oligos - not completely
> : deprotected may be? Funny thing is that the ligation worked - I could
> : see the ladder of the insert self-ligation. As if the DNA did not
> : replicate in E. coli.... Deprotection is simple (but stinky) - just
> : heat the oligo in ammonia for a while - you probbably know it better, as
> : you make the oligos yourself.
> We are high throughput so we're talking about several thousand oligos
> that are targeted to specific genes. Each PCR reaction is for a different
> gene--sometimes from cDNA templates or first strand from RNA. We PCR and
> (attempt to) clone several 96-well plates a week. I would die if every
> single synthesis I have done was bad. They are deprotected overnight in
> ammonium hydroxide at 55 and then dried down in the Speedvac.
> Not all the genes that are targeted clone the first time but most will PCR.
> The ones that don't clone correctly are the ones we battle. Just can't
> figure out why some worked in the past and others refuse to.
> Before any of this was a problem, I sequenced several PCR products to the
> ends and everything was where it should be. Of course this is a mixed population
> of possibly shorter 5' oligo ends (since not all oligos synthesize completely).
> There is an extra 6 bases upstream of the Sfi sites on the primers so digestion
> is more efficient than if just at the ends.
> I think what I'll have to do is TOPO clone some products before digestion
> and make sure a) it works b) the ends are complete.
> Thanks for any more ideas, Paul
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