Oligos for sequencing...

rupp at embl-heidelberg.de rupp at embl-heidelberg.de
Fri Nov 13 05:43:18 EST 1992


In article <11NOV92.12163090 at wums.wustl.edu>, wetsel_r at wums.wustl.edu writes:
> Hi Netters:
> 
> This may be a common theme or even an FAQ, but since I don't know I'll
> ask anyway.  To those that make their own oligos and do double stranded
> sequencing, what protocols do  you use, if any, in processing your oligo
> after it has been deprotected?  EtOH PPTs? Phenol?
> 
> David
> haviland at kids.wustl.edu

ReHi Netters,
I'm involved here at EMBL in the DNA synthesis/sequencing service and in
my hands a simple n-Butanole precipitation does the job after the 
synthesis:

Dry oligo down after deprotection o/n in a speedvac, dissolve it in
90 ul of water, add 1000 ul n-Butanole, vortex vigorously and spin
it down in a table top centrifuge.
You'll get a nice pellet which has to be "speedvaced" again to 
remove all the n-ButOH (would inhibit the enzyme!).

The above mentioned procedure precipitates almost 100% of the oligo 
whereas by EtOH purification you might loose some of your product.
It was adapted from:

                   M.Sawadogo, M.W.VanDyke
                   NAR 1991 Vol19, No3, p 674

and slightly changed.

Fell free to contact me for further information,

Thomas

RUPP at EMBL-Heidelberg.de



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