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:

Paul:

>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
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?

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
probing.

>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
 
=============================
 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 imm2.imm.uth.tmc.edu" 
 Voice: 713.500.2413  FAX: 713.500.2424
 ------------------------------------------------------  
Never take life seriously. Nobody gets out alive anyway.
=============================



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