Penny M Avoles
avoles at prairie.NoDak.edu
Thu Apr 13 16:01:36 EST 2000
What is the composition of your seqeunce you are
amplifying? Could secondary structure be forming? Try heating your
sample to 65oC before running to break secondary structure. If it is a
heteroduplex you could try to remove it using Mung Bean nuclease or SI
nuclease. I tried this procedure to identify RAPDs that formed
heteroduplexes. Unfortunatly, I never got it to work in my hands. You
might have more luck.
On Thu, 13 Apr 2000, Chris Garside wrote:
> Hi, I am currently trying to set up a competitive rt-pcr.
> I have transcribed two RNA standards.
> One of these standards has a deletion in it so that it can be differentiated
> from the full-length transcript on gel electrophoresis.
> Both standards amplify up on their own, giving a nice single band.
> However, when I amplify them up together, I get the appearance of a third
> band, between the two other bands.
> Does anyone know how to get rid of this band?
> Is it a heteroduplex?
> Will it mean that I can't use this for quantification of mRNA?
> ANy help or suggestions would be greatly appreciated.
> Chris Garside
> garside at zoo.utoronto.ca
> Chris Garside Telephone:416-978-3517
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