Help: Blotting DNA from acrylamide gel
smerage at cmg.health.ufl.edu
Tue Jul 18 12:05:43 EST 1995
I have transfered DNA from acrylamide gels in two ways.
The easiest is what we call a vacuum blot. See Lopez, et.al. 1993,
Biotechniques. 15:362-363. Essentially this involves lifting the gel off
the glass plate using the membrane you wish to which you wish to transfer
the DNA. This is placed on a gel drier. As the vacuum pulls the liquid
out of the gel it carries the DNA with it onto the membrane.
I have also used a semi-dry electroblotter. A o.5X TBE gel would be
transfered for 60 min. at 212 mA. This also worked very well.
In article <173DF8842S86.SBROWN at GAES.GRIFFIN.PEACHNET.EDU>,
SBROWN at GAES.GRIFFIN.PEACHNET.EDU (Stuart Brown) wrote:
> This is a real stumper for me. I need to make a Southern blot of some
> PCR products after separating them by size in a polyacrylamide gel. I
> need to use acrylamide in order to resolve small size differences between
> products (2bp SSR alleles) and I need to probe in order to verify that
> my PCR products really contain the repeats that I'm supposed to be
> amplfying. According to "Current Protocols" you can't use simple
> capillary transfer on polyacrylamide, they reccomend electroblotting.
> I found an electroblotter (Pharmacia "Multiphor II") and gave it a try
> using my "best guess" conditions (0.5X TBE buffer, 200 mA, 20 min) with
> absolutely no useful result (ie. blank film after probing filter and
> long exposure). I called the Pharmacia tech help line and was told that
> they don't have a reccomended protocol for blotting DNA with their machine
> because nothing that they have tried worked very well. Come on, this has
> got to be possible!
> Does anyone out there have any experience blotting DNA out of acrylamide?
> No reasonable suggestions sneered at.
> Cheers - Stuart Brown
> Stuart Brown | Plant Genetic Resources
> | Georgia Experiment Station
> INTERNET: | 1109 Experiment Street
> SBROWN at GAES.GRIFFIN.PEACHNET.EDU | Griffin, Georgia 30223-1797 USA
> BITNET: SBROWN at GRIFFIN | Fax: 404-229-3323
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