Help! Problem about Southern Blotting

David L. Haviland, Ph.D. dhavilan at IMM2.IMM.UTH.TMC.EDU
Mon Jul 21 10:16:40 EST 1997

At 12:14 7/18/97 -0700, Paul N Hengen wrote:


>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.

::Blush::  Goes without saying....  We stratalink the N+ or bake it 80'C
for 2 hours.  We have found that no membrane is "probe-able" if left in the
oven overnight by mistake.

>> 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
>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?

Hard to say, I've never used the Semi-dry system.  When I started in
Mole-bio with my post-doc, the lab had (that very month) changed from
sponges to the stones.  All I heard were nightmares about the sponges.  So
I'm afraid I was born, bred, raised on the stones...

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

The way my day goes, the gels are usually being set up to blot at about
4:00 pm <G>.  Overnight gels are usually run so slowly (genomic southerns)
that they aren't ready for blotting 'til about 2:00 and then they have to
go through the depurination anyway.  So they usually go overnight (14-18
hours - on average).

>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). 

I would think so.  They "hit" the membrane already denatured, ready for

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

No comment.

>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.

Ditto.  I'm afraid I've not experienced this.  When I pull my southerns and
northerns apart, only the membrane has various dyes embedded in it.
Virtually none in the gel and I've not noted any B_Phenol_Blue leaching to
the Whatman above.  yes, this is a vertical upward transfer.

If you're interested, Altec Biolabs, 116B Street, Boston, MA 02127
#617.269.1400.  No affiliation, just a very happy camper!  For northerns,
check out Virca et al Biotechniques 8:370-371.  Not to beat my own drum, we
have obtained and published results equal to those depicted in the
Biotechniques article.

Hope this helps,
 David L. Haviland, Ph.D.
 Asst. Prof. Immunology 
 University of Texas - Houston, H.S.C.
 Institute of Molecular Medicine  
 2121 W. Holcombe Blvd.  
 Houston, TX  77030 
 Internet:"dhavilan at" 
 Voice: 713.500.2413  FAX: 713.500.2424
Never take life seriously. Nobody gets out alive anyway.

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