How to re-amplify the DDRT-PCR band?

Gerd Nilsen gerdn at ibg.uit.no
Fri Mar 21 04:42:13 EST 1997


> Dear netters,
>
> I pleased my boss by good result of Differential Display. But I cannot
> re-amplify the band I interest in.

Here's the protocol we use: (Maniatis 6.46) The crush and soak
technique.
Make elution buffer:  Ammonium acetate 0.5 M, Mg acetate 10 mM, EDTA
1mM,
SDS 0.1%.  Excise gel piece, crush it in 50-100 ul of elution buffer.
Incubate at 37C for 4 hrs, then centrifuge 12,000g x 1 min, recover
supernatant.  Add another 50 ul of elution buffer to pellet, votex,
centrifuge, combine supernatants.  Filter the elution through glass wool
to remove large particles, then Etoh precipitate and wash.  Bring up in
a
small volume (10 ul) of TE Buffer.  Re-amplify using the same PCR
conditions and primers as DD-PCR.

Good luck,
Louis Alarcon
U of Pittsburgh

Looks like a good protocol, to obtain even better recovery during
presipitation, you could add a few microlitres of 10% glycogen. It don¥t
hurt the re-PCR.





More information about the Methods mailing list