Technique / Molecular Biology / DNA analysis techniques / DNA southern blot


Southern blot background



Southern blot background

Some common causes for high background in southern blot hybridization:

1. High concentration of probe (should not exceed 5-10ng/ml). Probe sequence too short (for oligonucleotide probes). Probes not denatured properly.
2. Low efficient purification of labeled probe to remove unincorporated nucleotide.
3. Hybridization buffer contains particles.
4. UV crosslinking or baking membranes with high salt.
5. Insufficient hybridization blocking.
6. Membrane dried during hybridization process.
7. Washing condition is not stringent. Low SDS concentration in washing buffer.
8. pre-hybridization is not sufficient.

Troubleshooting discussion on high background in southern blot hybridization :

I have been having background problems with the top half of my southern
blot. The bottom half of the gel is devoid of any background, however
the top is completely black. I ran a second the gel without EtBr,
thinking that may be the problem but I obtained the same background
pattern. I am using a positively charged nylon membrane, an 800bp P32
probe, and I hybridize and wash at 55C. Has anyone else experienced
this background problem? Any help woud be greatly appreciated. In
addition, has anyone prepped genomic DNA from Aedes aegypti and
succesfully used it for a Southern. If so what protocol(s) did you use.
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I trust you have excluded the possibility of a light leak?
==========================================
Something like this happened to me before. Unfortunately, I got two
different suggestions, and I changed both parameters at once, so I dont
really know which one made the difference. First, I thoroughly cleaned the
gel box, because it was suggested that there was DNA in the box/buffer and
that was running into the gel in a uniform manner and infiltrating the whole
gel, and subsequently showing up on the membrane. Secondly, I was
hybridizing in a roller bottle, and portions of the membrane were
overlapping. It was suggested that I was getting an air pocket that was
preventing proper washing of the membrane, so I switched to a larger bottle
where I would minimize the overlap. This combination worked for me, I hope
it helps you (although I dont know if you are using a roller bottle).


Brian
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You didnt say, but I will guess your using a roller bottle and the
overlap is causing the noise.


Switch to seal-a-meal bags. They are cheaper and you can throw the
whole mess away after the experiment. People in my lab started using
the bottles against my advice and switched back to the bags when the
notice my blots were always cleaner.


Regards,
John Thompson
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Hi Michael and netters,
we are using roller-bottles and had the same problems. The overlap should be
no problem, as we used to get clean blots. We changed the dextran-sulfate in
the hybridization solution to Bio-mol and it was o.k..
Hope that is useful info
Frauke
==========================================
you may also want to heat up your washes if you dont already
and if you are using a roller bottle, MAKE SURE THE MEMBRANES ARE
ACTUALLY ROLLING INSIDE
if not, rerole them and make sure they are nice and tight
that worked for me
==========================================

Last update 03-Oct-2000, Rating of 2 votes.

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By shown on 09-Sep-2008
I have been using roller-bottles for more than 3 months. I usually put two membranes in one hybridization tube. The big problem is that the background of the first membrane exposed directly to the hybridization solution was so high that I could not tell the bands, but the second membrane, which was covered by first membrane and attached to the wall of tube, was good and the background was normal. I do not understand it. Could someone give me advice?

Thank you very much!
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By viqar on 17-Jan-2005
HI ! thanks a lot this question and the answer to this have brought an end to a similar problem i have been facing in my lab.
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By biowww on 03-Oct-2000
You can add your comments here.
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