Primer preparation

Marianna Max drmax at casbah.acns.nwu.edu
Fri Aug 27 21:35:47 EST 1993


In article <cooperj.3.746483203 at nhlbi.nih.gov> cooperj at nhlbi.nih.gov (John Cooper) 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?
>
>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


I sit the bottle of primer on top of the 95 degree hot block for 2hr. Then
I take an aliquot (usually 300ul) and ETOH percipatate it with 1/10 vol 3M
NaAc and 1ml of 100% etoh. Wash the pellet 2x with 70% let air dry and add
back 300ul of water. From our machine this makes an oligo concentration
that is just about right for PCR. The initial pellet is sometimes whitish
but it becomes clear after the 70% etoh wash.

Max



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