cleaning PCR primers

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Mon Dec 12 15:05:44 EST 1994


Jo Caine writes:

> We have just made some oligos for PCR on a 50pmol scale, cleaved them from 
> the column, deprotected and dried them down.  When they are redissolved in 
> water there is visible rubbish in the tube, which does spin down.  I have 
> tried using the clear supernatant, diluted, in a PCR reaction and get 
> nothing.  My question is, does anyone know if dirty primers can inhibit the 
> PCR reaction?  And if so what is the best method (and simplest) for 
> cleaning them up? 

The stuff you see is the blocking groups that were cleaved off but not 
physically removed.  I don't know if it causes any specific PCR problem,
but I have seen it generally cause trouble with drying, resuspension, and
quantitation of the oligos.  We get rid of it by passing the deblocking
mix (still in ammonium hydroxide) through a C18 cartridge before drying.
The blocking groups stick on the resin, while the oligo goes straight 
through.

Steve Hardies, Dept. of Biochemistry, Univ. of Texas HSC at San Antonio
Hardies at thorin.uthscsa.edu
 




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