cleaning PCR primers

David R. Rockhold drr at PW.USDA.GOV
Mon Dec 12 13:53:51 EST 1994


We've found that simple ethanol precipitation after drying down produces
much cleaner oligos.  We just centrifuge the oligo after resuspending, and
add 1/10 vol NaOAc and 2 volumes of EtOH to the supe.
>
>>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?>Jo Caine
>
>We received some commercially prepared primers in a similar state, which did 
>not work properly. The issue was not resolved entirely, but seemed to be 
>due to material coming off the column. The material from the column 
>resulted in a spuriously high OD value, resulting in over dilution of the 
>primers (i.e. it was difficult to know how much primer we had), rather than 
>the dirty primers interfering with PCR. 
>
>




More information about the Methods mailing list