Differential Display PCRs - help needed!

Louis Alarcon lha at med.pitt.edu
Thu Mar 28 13:21:25 EST 1996


In article <4jc14k$htt at mercury.hgmp.mrc.ac.uk>, jcameron at hgmp.mrc.ac.uk wrote:

> I am currently having terrible trouble remamplifying cDNAs that I have
cut out from
> differential display gels.....
> 
>
> Jessie Cameron
> Galton Laboratory
> Univesity College London, UK

We had similar problems until we used the "crush and soak" method
(Maniatis 6.46).  In brief:  crush the gel piece and mix with 1-2 vol of
an elution buffer
wich consists of: Ammonium acetate 0.5M, Mg acetate 10 mM, EDTA 1 mM (ph
8) and SDS 1%.
Incubate at 37C for 3-4 hours (longer for oligos > 500 bp). Spin 12000 g 1
min, recover supernatant and save.  Mix pellet with 1/2 vol of the elution
buffer, voretx, spin as above and combine supernatants.  Filter the
supernatant through siliconized glass wool, then etoh precipate. Bring up
in desired volume of TE buffer (we use 10 ul) and re-amplify using 2 ul of
this sample.
This method works well for isolating cDNA from diferential display.

Lou Alarcon 
Dept of Surgery
U of Pittsburgh
lha at med.pitt.edu



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