I hate PCR

taylorpm at ARIEL.UCS.UNIMELB.EDU.AU taylorpm at ARIEL.UCS.UNIMELB.EDU.AU
Sun Sep 8 18:59:18 EST 1996


Hi everyone,

I am havong difficulty with some PCR, just for a change.

I am trying to amplify a region of DNA encoding 16S rRNA. My primers are
well known, in our lab at least. They are 1525f and 25r. Previosly, these
have produced lovely bands of the right size, approx. 1.5 kb, at fairly
typical temperature settings and cycle lengths. This year,  I am trying to
do the same thing and am having no end of difficulties.

The product is a smudge that has a more concentrated area at 1.5 kb, but
also has lots of larger and smaller products, ranging from about 2.0 to 1.0
kb. The larger products rule out a degradadation problem.
I have tried increasing the  annealing temperature by about 10 degrees C (
to 55) but I still get smudgy products. Does anyone out there have any
suggestions? What could be going on here??

Thank you for your time,

Paul

-----------------------------------------------------------------

Paul Taylor
Department of Microbiology
University of Melbourne
Parkville   Victoria    3052
AUSTRALIA
Ph  : +61 3 9344 5698
email : taylorpm at ariel.ucs.unimelb.edu.au





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