RT-PCR-Multiple bands-unusual problem
Simon.Dawson at nott.ac.uk
Tue Jun 25 12:41:52 EST 1996
Paul Mozdziak wrote:
> interest. The probe samples amplify beautifully. However, when I run
> my rt-pcr stuff from muscle out on a 3% gel (sigma agarose (50-1000
> bp) I get two bands (one at 150 bp--what I should get and one a few base
> pairs smaller)--they just barely resolve. I ran these samples out
> shortly after I finished the pcr.
> Any ideas to explain my problem and too solve it?
Are you doing the rt-pcr with both the cDNA control template and the
muscle RNA together? If you are and the cDNA control is a deleted form of the
sample you're trying to measure, what you are seeing might be due to the
formation of heteroduplexes which will run with a slightly different mobility.
I had a similar problem with some competitive rt-pcr I was doing (I could see
3 bands - the 2 I wanted and a third band that ran close to the larger
expected product) and I had to label the products with P32 and separate them
using denaturing acrylamide electrophoresis.
Hope thats of some use. Drop me a line if you want anymore details.
Dr. Simon Dawson TEL:+44 (0)115 9249924 ex. 44787
Dept. of Biochemistry FAX:+44 (0)115 9422225
Queens Medical Centre Email:Simon.Dawson at .nott.ac.uk
Clifton Boulevard http://www.ccc.nottingham.ac.uk/~mbzspd/Simon.html
U.K. "Back off man, I'm a scientist!" - Bill Murray.
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