Help! Problem about Southern Blotting

Paul N Hengen pnh at ncifcrf.gov
Fri Jul 18 14:14:25 EST 1997


David L. Haviland wrote:

| I have found a problem in the Southern Blotting. All digested
| genomic DNA (size from 11kb to 0.5kb) and lambda DNA marker still stayed
| in the agarose gel after overnight capillary transfer. While the
| bromopheonl blue dye have successfully transfered to the membrane.
|
| I have used 10X SSC as transfer buffer and the treatment of the
| gel was the incubation with agitation 0.4N NaOH with 0.6M NaCl for 30
| minutes and then 0.5M Tris-HCl pH 7.6 with 1.5M NaCl for another 30
| minutes. The methods have been used in our lab without any problems. I
| even my boss don't know what wrong in the blotting. Please help me to
| solve the problem.

Don't forget to UV cross-link! It can result in loss of signal if
little DNA is transferred. Turn the membrane upside-down onto a 254
nm UV transilluminator for 10 min. or blast it with a UV Strata-linker
or Hoefer UV X-linker.

> I would strongly recommend the Altec blotting stones. Compared
> to using sponges and the like, we have had spectacular results using the
> Altec Blotting stones (Altec Biolabs, 116B Street, Boston, MA 02127
> #617.269.1400)

Thanks David! I am having a similar problem when transferring DNA from a 8.0%
polyacrylamide gel onto Pall BioDyne A membrane. I am using 0.3x TBE buffer for
a semi-dry transfer using a Hoefer TE-70 Semi-Phor system.  The problem is that
when I run the transfer at 30 mA for 30 min., the DNA hardly transfers from the
gel, as seen by little transfer of loading dye (Amaranth) from the gel to the
membrane and low signal after development of the membrane. If I run at 30 mA
for 40 min., I see the dye pass right through the membrane into the blotting
paper and blobs of some dye left on the membrane. Development from this kind of
transfer results in spotty bands on the film later. This has been quite
variable and I am having a heck of a time getting an in-between-er within the
10 min. time window, which in the past has given me great results.

1. How does the stone system differ WRT the Semi-dry system?

2. How long should I transfer with the stone method?

3. Do you think I would get better results by changing my transfer
solution from 0.3xTBE to 0.4 N NaOH? (DNA fragments are in the 20-50
bp range). 

4. What conditions do other people use when blotting with the TE-70 unit?

5. Are there any cool tricks for trapping the DNA onto the membrane?
I suspect the blobs are coming from the DNA passing right through the
membrane and onto the semi-dry blotting paper behind it.

-Paul.

--
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* Paul N. Hengen, Ph.D.                           /--------------------------/*
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