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...
>>Picard at helix.nih.gov
I have been running our ABI DNA synthesizer for a couple of years now.
First of all, do not buy ABI phosphoramidites as they charge you an arm and
a leg. You can pick up phosphoramidites through Fisher, from a company
called Cruachem. These tend to give a higher yield.
Anyhow, you can shorten the deprotection time by addition of a few ml of
ammonium hydroxide to the deprotection vial, or you can use FOD
phosphoramidites. These are an alternative to the cyano-ethyl (CE)
phosphoramidites, and deprotect in an hour. These you can purchase from ABI
at $65 per 0.5g. With institutional discount you may be able to pick these
up from Fisher at a quarter of the cost, as well as the usual CE type.
The extra crud, well I call this sand. I suspect that either it is some
insoluble precipitate (how did I work this one out?) which is a by-product
of the cleavage from the column - or it is just in fact glass-powder
generated by the CPG beads.
Hope this may help
leach at mbcrr.harvard.edu