PCR....help

Daniel Gene Sinclair danielg at earl.med.unc.edu
Mon Jan 29 11:20:14 EST 1990


In article <90Jan26.085914est.57547 at ugw.utcs.utoronto.ca> FS300512 at yusol.bitnet writes:
>
>Recently we are experiencing problems with our PCR reactions.
>One day the reactions work fine, the next day, if we repeat the same
>reactions (everything the same) they just don't work. It does not

Is your dna properly resuspended?  We have not had this interfere with
pcr, but in doing southerns, non-homogenous dna suspensions cause uneven
amts. of dna to be put into each lane.  If you are using genomic dna, we
have found (with our amplimers) that even a 10 fold reduction in dna will
still produce readable bands on a gel (i.e. a reduction from 1 ug to .1
ug) in pcr.

Also, we add only .5 uL of taq polymerase using a 20 uL pipetman - not a
safe thing to do, if you don't watch carefully, you may not include any
enzyme.

Simple suggestions, I know, but sometimes that's the thing that is missed.

daniel g. sinclair
University of NC, Chapel Hill



 Disclaimer: My opinion means | 'If you only knew how much I was holding
             nothing, but His |  back, you would commend me'
             means everything.|      - Charles Spurgeon, 19th century
                              |        evangelist (on humor in preaching)



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