gel smear without stop

MELANSOND melansond at am.abru.cg.com
Thu Dec 12 08:32:16 EST 1996


***Hi:
***
***   I had no too much experience in pcr, Recently I try PCR and run gels
***that showed  smear without distinct stop and no visible band. I check the
***book for pcr that you must optimization experiments.Only change that is
***difference from bewfore is using Taq from PE company.  How can I do ?
***  the following the my methods
***
***10x pcr buffer( P.E company)   5 ul
***MgCl2(25mM)                    3ul
***DNTP(2.5mM)                    2.5ul
***5' primer( 20uM)               2.5 ul
***3' primer(20um)                2.5ul
***H2O                            32.5 ul
***Taq( 5U/ul, PE company)        0.3ul
***CDNA temple                     2ul
***
***
***
***
***S-J
***
This question is hard to answer without more information.  What is your
template, is it simple or complex? How were your primers designed? How were the
reaction conditions determined? As far as the source of taq, this can and has
had an effect. However, silly question, before you use the taq, do you vortex
and spin down to ensure complete mixing of the enzyme in the glycerol? Smears
sound a lot like non-specific amplification, too much enzyme, too low annealing
temperature. Determine the Tm of the primers and subtract 5 degrees, this
should be your annealing temperature.
Good luck




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