rover at ns1.livnet.com
Fri Dec 1 20:34:43 EST 1995
>I have been doing differential display reactions and get good gels and can
>even amplify the bands out of the gel , But when I do northerns with these
>reamplified bands there isn't any signal at all on the blots. I know my
>RNA is fine and the transfer is fine too. Can't figure out what the
>Any help would be really appreciated.
>apotnis at aim.salk.edu
How many amplified bands have you isolated?
One thing you may want to take into consideration is that differential
display is an amplification technique. The differentials that you are
picking up may not be sufficiently expressed for you to detect them on
Northern analysis. You could try using some more sensitive techniques
such as sequencing your product followed by quantitive reverse
transccription and PCR. The fact that you have bands demonstrates that
you are amplifying something though.
The technique does work, we have isolated a number of differentials
and substantiated it with data from Northerns. The probes for our
Northerns were obtained from precipitating differential bands in the
presence of glycogen, reamplification, isolation from 5% page ,and
radiolabeliing using the random priming method.
We have an article coming out in FEBS letters in the next couple of
months in which we used the technique, and there are many other
references out there.
hope this helps
--Rover The Mad Molecular Biologist
rover at livnet.com
barlow at picard.evms.edu
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