cleaning PCR primers

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 

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

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