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Primer preparation

Bob DeLisio delisior at rnisd0.DNET.roche.com
Fri Aug 27 16:21:23 EST 1993


"cooperj at nhlbi.nih.gov" writes:

>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?


You can either deprotect at 65c for 3 hours or even at 85c for 1 hour. However
you increase the risk of the vials exploding ( which fortunately has never
happened to me!). I use Wheaton vials and caps but you may want to test the
higher temperatures on your own vials first.


>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?        


I usually just spin out any crud in an eppendorf for 5-10 minutes. The crud
does increase the OD260 readings and should be removed prior to quantitation.

>Any insights will help, thanks...  

Good Luck
Bob



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