Discuss Southern Blotting - (Jul/28/2005 )

Usually, when We run gel before southern blotting, the low voltage will be used, say, 1-2 V/ cm. what if the high voltage and low temperature will be used? This is simliar to fingerprinting gel. I am wondering if the blotting result will be getting better. suggestions, ideas will be very welcome!

All you want from your run conditions is good resolution of the DNA fragments. The only complication with southerns is you are generally loading quite a bit of DNA into each lane and high amounts of DNA can cause smearing.

What I have done is start of the gel at a low voltage and run it for 30 minutes to get an initial separation of the fragments and then turn the voltage up.

Thank you,Daniel.

Usually, I load much less digested BAC DNA than genomic DNA. But based on most protocols, 50 ul (~10 ug genomic DNA,total) will be used for one lane, I also think that it is too much. However, if you load a little bit of DNA, do u think that some bands will not be visible? One more thing, you said, you will use high voltage for the later running. Is that working well? Does the high voltage increase the temperature of buffer and then make the bands not sharp? I am supposing that you do this at RT.

If you use less DNA then you will be able to run it faster. The downside is if you half the DNA then you will need to double the expose time. It has been awhile since I have done a southern (I personally hate them), but my approach seemed to work fine last time I did it. A higher voltage will increase the buffer temp - if you are worried you can run always run it in cold.

Prepare for a headache if you're using a non-radioactive labelling/detection kit. I've been troubleshooting this thing since April..

But yeah, all that really matters is that you run it slow enough for good resolution. Obviously if you're running a ton of DNA then you will want to run it slow. You're asking about running it at a higher voltage in a cold room or something to speed the process up? I had a professor that absolutely HATED waiting for gels to run. He'd set up a gel box on ice and run the thing at 200 mV. Of course, this wan't for genomic DNA.

-Hank

try with "borax"

i have never tested this buffer in Southern blotting experiment but it works very well in my DNA electrophoresis:

Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis.
Brody JR, Kern SE.
Johns Hopkins University School of Medicine, Baltimore, MD, USA.
PMID: 14989083


simply make the gel and electrophoresis with:

sodium boric acid 10 mM final
EDTA 1 mM
ethidium bromide 1 mg/l

and nothing else!

you could enhance the voltage safely...

Seb_

Good suggestion Seb

First of all, thanks Seb for sharing the information with us here. One question is that SB buffer is being used in your lab, but you have no idea about the effect on Southern or Northern blotting, how come? Nobody in your lab conducted blotting with this buffer?

i have always used tris-acetate for electrophoresis (agarose) before alkali blotting of DNA...
since i have no more oppotunity to carry out Southern blot, i let you try with sodium boric acid ;-)
Seb_