northern blot help needed

elader at ambion.com elader at ambion.com
Thu Nov 6 18:51:10 EST 1997


In article <63ss6h$jbo at whitbeck.ncl.ac.uk>,
  laura gray <laura.gray at ncl.ac.uk> wrote:
>
> I am using Capillary blot method for northern blotting as in Maniatis.
> Recently about 50% of the RNA has not been transferring to the membrane (
> Genescreen plus). i have tried new batches of buffer and agarose to no
> avail. Can anyone help?

A couple of thoughts. First, I think it is unlikely that the agarose and
buffer are to blame unless you are making a higher percentage gel all of
a sudden. Are you making your gels thicker than you had been? This will
have an effect. Do you have EtBr in the gel? Are you staining it
afterwards. We find that you must limit the EtBr you use. The preferred
method is to put 10 ug/ml EtBr in the GEL LOADING SOLUTION. The etbr will
electrophoresis out of the top of the gel , yielding a gel with no etbr
background. Do not use more etbr, it'll knock down the hyb signal.

The membrane you are using is a good one. DO not change brands. Limit the
thickness of the gel to 6-8 millimeters. Yes, I know it's thin, but it
helps. Be careful not to use a huge weight on the top of the stack. This
compresses the gel even more than usual (which sucks to start with). Try
an upside down transfer (see the ambion.com web site), it allows you to
use a very small weight and does not compress the gel -> better flow
throughthe gel.

Want to chat? give me an e-mail or call. It is toll free from england
0800-96-8292. I promise I won't try to sell you anything.

Eric    elader at ambion.com

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