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Primer preparation

alan shore acpub.duke.edu
Mon Aug 30 07:28:56 EST 1993


In article <cooperj.3.746483203 at nhlbi.nih.gov>, cooperj at nhlbi.nih.gov (John
Cooper) wrote:
> 
> Any body have a good protocol for preparing primers after synthesis. We have 
> an ABI DNA synthesizer which we use to make our primers. We deprotect them 
> by incubating at 55c for 5 hours then dry in a savant spinvac. My questions:
> 
> 1) Is there any way to speed up the deprotect process?
> 
> 2) After I dry my primers there's always a bunch of crud at the booton of 
> the tube that never solubilizes when I add h20. Is this wasted oligomer? Is 
> there a way to avoid this?
> 
> Any insights will help, thanks.


I use an  80 degree C  heat block to deprotect my oligos,  I place the dna
in the heat block for at least 30 min and try to leave it no longer than 1
hour.  If I do a trityl ON  synthesis,  I add 70 uL of triethylamine,  this
is supposed to stabilize the resuspended product.   I also use a heat block
with a gas manifold on top sold  by Pierce called a reaci-vap,   I blow
house compressed air over the top of the deprotected oligos,  this heat
block is at 80 C also,  and  in 30 min,  I have a dried oligo. I do this in
a hood.

I think the dried  insoluble crud is a sometimes by product of the
deprotection,  but  have no proof.  It could also be something fishy in a
valve block,  since it is  a sporadic but not uncommon  event at  my place.

Three  cheers to all you folks who said you get better results with
everyone other than ABI.   I  wish I could tell you what my ABI rep said
about their concerns about quality, but I don't have it on tape and I can't
 afford a good lawyer.

Alan Shore    dnamaker at acpub.duke.edu   moving to Cincy soon



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