Non-radioactive gel shift

hap hapNOhaSPAM at brew-master.com.invalid
Wed Mar 8 07:21:15 EST 2000


In article <8a4hov$bvl$1 at kestrel.csrv.uidaho.edu>, "Don
Walthers" <gene_jockey26 at yahoo.com> wrote:
>I'm using biotinylated oligos to PCR my protein-binding site. I
then run the
>samples on an polyacrylamide gel. I have access to an ECL kit
which has
>whatever enzyme conjugated to avidin. Does anyone have any
experience
>treating the gel directly with the enzyme/substrate solution
then slapping a
>piece of saran wrap between the film and gel or is blotting
necessary. If
>so, any recommendations on what to transfer the DNA to? It's
been a while
>since I've done this and it was with proteins anyway. Are the
membranes the
>same?
>Thanks for any help,
>
>--
>Don Walthers
>B.S. Microbiology - U of Illinois at U-C, 1996
>Ph.D. Microbiology - U of Idaho, 2000?
>gene_jockey26 at yahoo.com
>
>
>
>

Don,
We do dig-labelled gel shifts, and we prefer to transfer the gel
to a positively charged membrane, and then do detections.  We
simply transfer the gel by capillarity, and get most of the DNA
transferred in 1-2 hours (you can also transfer overnight, as
well).The DNA stays stuck to the membranes pretty well, and UV
crosslinking is not necessary, though it does improve the signal.
In our experience, any positively charged membrane is okay - we
are currently using NEN genescreen plus.

Hap



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