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Primer preparation

Yan Tony Lee ytony at acs.bu.edu
Fri Aug 27 13:24:41 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...
> 
> Cooper
> 
> Picard at helix.nih.gov


Hi,
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



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