RT-PCR-Multiple bands-unusual problem

[Simon Dawson]

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?
> 

Hi Paul,
        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.

         Simon.

-- 

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
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