10 replies to this topic

[GradMom]

Hi Everybody!
My first-ever Southern blot is giving me a headache.  After an overnight exposure, I got an extremely faint band in my control lane (with my probe fragment), nothing in my marker lane, and only the faintest of bands in two of my sample lanes.  This exposure had relatively high background on half of the blot (I assumed the background had something to do with the way the blot was arranged in the hybridization tube).  I stripped the blot and re-probed.  Alas, I got the same faint band pattern.  I'm following a protocol that my lab has used before, I am using solutions that have been used successfully before (I checked the pHs just to be sure), we checked the dATP we used and it incorporates well.  Here's a rundown of what I'm doing:

Digest Genomic DNA overnight (I'm using enzymes I've used before and they are cutting the DNA)
Run on a 0.7% TAE gel (I check the gel on the UV-light box and everything seems fine so far)
Flip the gel (We're in a dry climate and supposedly this helps)
Depurination 15 minutes (dyes change color)
Denauturation 30 minutes (dyes more or less change back)
Neutralization 30 minutes
Assemble transfer apparatus (we use GeneScreen membranes which have never been a problem before)
Transfer overnight
UV-irradiate 12 minutes
Prehyb- 65 degrees 30 minutes
Add the radiolabeled probe
Hybridize overnight at 65 degrees
Rinse w/ 3X SSCP, 0.1% SDS 3 times at room temp 10 minutes
Rinse once w/ 65 degree 3X SSCP, 0.1% SDS 10 minutes
Rinse once w/ 65 degree 0.1X SSCP, 0.1% SDS 10 minutes
Expose

I'm currently running some Plasmid DNA for a quick and dirty check of my conditions.  The enzymes have cut to completion and the dyes in the gel changed colors.  I tried to visualize the DNA after the Depurination step and I couldn't see anything. Does anyone know if the Depurination step causes the ethidium bromide to dissociate from the DNA?  Or has my DNA gotten away from me somewhere?  Any other suggestions?

Please help!   I thought Southerns were supposed to be the easy ones?!

[perneseblue]

I assume that your ladder is radioactive.

If so, than the lack of ladder once the x-ray film/ phosphoimager cassette indicates no DNA on the membrane.

No DNA on the membranes means
1 - the transfer of DNA from gel to membrane was poor
2 - the fixing of DNA to membrane was poor
3 - really bad gel where all the DNA was degraded.

(1) is most probably under the assumption that the marker is radioactive. Do you have anybody to show you how to prepare the southern blot sandwich? You need to make the interface between gel and membrane water free. My lab uses a ruler to wipe off the layer of water/buffer from the gel. And remove any excess buffer from the southern blot membrane, before overlaying it on the gel.  

It is also possible that the pile of blotting paper used to draw buffer through the agarose gel, came in contact with the felt cloth surface (which the gel rest on.). If so this could short circuit the southern blot apparatus as buffer can be drawn directly from the felt cloth bypassing the agarose gel.

I also have a question. How big is the band you are probing for? depurination is only used if you are looking for bands 15kb and above.

[GradMom]

My ladder is not radioactive.  I ran lambda DNA cut with HindIII and hybridized with both probes.  I can't think of any reason why the gel would be bad.

My P.I. set up the first transfer to show me how to do it.  We surrounded the gel with plastic wrap to keep the buffer from bypassing the agarose gel.  Another thing I didn't mention, the dye from the gel transfers to the membrane properly.  The first few pieces of blotting paper have traces of dye.
  
I'm probing for a 2.5kb fragment so I could skip the depurination step.  Most protocols I've looked at say that depurination is optional.  I took this to mean that it shouldn't hurt.  If this next transfer doesn't work, I'll try skipping depurination.

[perneseblue]

yes, you should skip the depurination. In this situation, depurination does not help as it removes bases, which in turn make the signal weaker as the probe has a harder time hybridising with the DNA.

Do you know the DNA sequence of your probes? Are they AT rich? Since the lambda ladder isn't radioactive, it is possible that your washing is too stringent. You could skip the high stringency wash (0.1X SSCP, 0.1% SDS), or lower the wash temperature.

Skip the depurination first. If that doesn't help, reprobe and start fiddling around with the washes.

[mdfenko]

12 minutes seems to be a long time for uv crosslinking, the dna will be fragmenting during the procedure and you may want to limit it.

i seem to remember a 15 to 30 second exposure is usually sufficient. the protocol given at the fermentas website calls for 2 minutes uv crosslinking.

you can also replace uv crosslinking with baking.

or you can use a charged (+) nylon membrane, in which case neither uv crosslinking nor baking is necessary.

[GradMom]

Thanks for all the advice.  I ran a smaller gel with some plasmid DNA and it worked fine.  It turns out that the gel I initially poured was just thicker than those which had been run by others using the same protocol.  I ran my genomic DNA samples on a thinner gel and the southern worked like a charm!

[perneseblue]

out of curiosity, what where the gel thickness used in the two attempts?..the attempt that failed and the attempt that worked.

[molgen]

Quote

Another thing I didn't mention, the dye from the gel transfers to the membrane properly. The first few pieces of blotting paper have traces of dye.

When I started Southern blotting (~10 years ago) we had a problem.
The smaller parts were going throw the membrane into the paper.
What we ended up doing was to irradiate the gel with UV (to break the long chains and then have a shorter transfer time.

[GradMom]

Failed attempt- 1.2 cm
Successful attempt- 0.8 cm

[perneseblue]

NMlabtech, on Feb 18 2009, 01:16 PM, said:

Failed attempt- 1.2 cm
Successful attempt- 0.8 cm

Thanks. It is good to know another factor that can influence a southern blot's success.

[shyam]

gud to know abt the above

Edited by shyam, 13 May 2009 - 06:12 AM.