Ligation problems

P.C. 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,

clone'em all!

Peter


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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