In article <cooperj.3.746483203 at nhlbi.nih.gov>, cooperj at nhlbi.nih.gov (John
>> 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
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