HEX-labelled primer doesn't work in PCR ?

Shirley Horn-Saban lvsaban at weizmann.weizmann.ac.il
Thu Mar 21 07:37:26 EST 1996


I have a problem with primers to which an ABI dye is attached (for
running on the ABI 672 GeneScanner).

My problem is as follows: We have a FAM-dyed oligo (blue) which works
great. Then we ordered from another company (BioServe) three additional
oligos, 1 FAM and 2 HEX-labelled (green). While the FAM works very nice,
with the other two I was unable to get a PCR product AT ALL (works good
with identical, but unlabelled primers).

Naturally our first thought was the primers are no good, and we
re-synthesized one of the HEX primers at ABI's, which is more expensive,
but the quality is guaranteed. This, as well, didn't work.

I tried different modifications, such as adding DMSO to PCR reaction, 
reducing Tm, using TaqPlus (Stratagene, highly processive), increasing
primer concentration and other manipulations, but with no success.

The PCR is done on human genomic DNA, purified in several different ways.

Has anyone else experienced similar problems and been able to work out a
solution? 

Please also reply by e-mail to:  lvsaban at weizmann.weizmann.ac.il


Shirley Horn-Saban
Department of Membrane Research and Biophysics                  
Weizmann Institute of Science
Rehovot, ISRAEL
tel:  +972 8 9342 455
fax: +972 8 9344 112                                                     
             e-mail: lvsaban at weizmann.weizmann.ac.il



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