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Southern blot help


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#1 prion78

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Posted 14 June 2010 - 10:35 AM

I am trying to detect a two copy gene using Roche DIG southern blot protocol. Our probe is 192bp long, it shows nice and strong on a gel and also shows the typical displacement when run next to an unlabeled probe. When I do the southern blot all I got is a smear in every lane. I've tried different concentrations of probe using 2ul of the PCR product, using 1/10 and 1/40 dilution of the PCR product and still get the same results (well...the smear goes progressively lighter when I use the dilutions but no bands so far). I am starting to think that my problem is the DNA digestion. I work with fish liver DNA, I generally use 1-5ug of DNA and 10-50 units of the restriction enzyme (EcoRI and BamHI), incubate it overnight at 37C. Now I'm trying a 6 hour digestion but seem to have the same problem.
These are the conditions of my experiment:

overnight digestion of 2ug of DNA with 20units of enzyme in a 30ul volume reaction
All product is run on a TAE 1X 0.8% agarose gel for 4hrs at 70V over ice (I'm not using EtBR anymore)
Gel is transferred overnight using capillary method with 20X SSC
Next day the membrane is air dry and fixed using UV crosslinker
membrane is prehybed for 3 hours with DIG easy hyb
and then hybridized with new easy hyb with the denatured probed


Can anyone help me with this ordeal?

frustrated postdoc

#2 phage434

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Posted 14 June 2010 - 01:28 PM

You have not said how your dna is prepared. An overnight digestion gives any lurking nucleases wide latitude to do nasty things. I see no reason to do it overnight. 30 minutes is probably more than adequate. 1 unit should cut 1 ug in an hour.

You can optimize your binding and visualization with dot blots rather than doing gels. Dilute serially your dna (cut or uncut) and dot it on a membrane, crosslink, and detect to determine your limiting detection and to optimize detection conditions. Only then run gels and blot.

#3 CatarinaCarvalho

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Posted 20 June 2010 - 11:58 AM

Hi!
I am trying to perform a Southern blot of mouse genomic DNA extracted from ES cells.
This is my protocol
1. Digest 7micrograms with my restriction enzyme (5U per microgram) o/n
2. Run a 0,7% agarose gel in TAE buffer, with gel red. 45V for 24h.
3. View the smear on Image master.
4. Denaturation (0,5M NaOH + 1,5 NaCl) 2 x 20min (the fragments that I want to see have 7kb and 9kb, so I donīt do depurination)
5. Neutralization (Tris-CL 0,5M + NaCl 1,5M, pH 7,5)
6. Transfer in a vaccum device using neutral transfer buffer (20x SSC) for 1h, to Hybond N+ membrane.
7. Cross link with UV
8. Pre-hib at 55C for 1h
9. Hib with myDNA probe (430bp) labeled with alkaline phosphatase (Alkphos direct labelling and detection kit from GE - Amersham) at 55C o/n.
10. Add substrate and wait 5h until photograpg the blot on storm.

I am having huge trouble! I only see the DNA smear from my digests! I though that It was from gel red, but I already discarded that hypothesis...I can't see a specific band. Already waited 24h for revelation and the smear just intensified!

Please help me!

Thanks.

#4 prion78

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Posted 29 June 2010 - 07:25 PM

The DNA is phenol/chlorophorom/isoamyl extracted from trout liver (protocol from Maniatis). I I did a test with different times of digestion the DNA was not completely digested (I did 1hr, 2hr, 4hr and 6hr). I did the dot blot but using a PCR product (serial dilution). To do it with the genomic DNA, should I digested first and then dot blot it? or can I do it with the undigested DNA?

#5 phage434

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Posted 30 June 2010 - 05:10 AM

The problem could easily be hybridization conditions rather than poor cutting. If you probe is non-selectively sticking to all of the DNA, you would see the smear. Have you tried reduced salt or higher temperature in your hybridization washes? It would help to know more precisely what your protocol for both the digestion and hybridization looked like. I'd like to know, e.g., what volume you are doing the digestions in, and what fraction of that volume is your isolated DNA. Residual phenol in your DNA could easily inhibit the restriction digests. You might try an extra chloroform extraction, followed by ethanol precipitation and two or more 70% EtOH washes.

#6 Tamara M

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Posted 16 November 2010 - 01:10 PM

It sounds as if your washes after hybridization are not stringent enough. Maybe you could try a ore stringent wash in 0.1x scc with higher temperature to reduce non-target binding. Moreover, you could reduce the amount of DNA you use to see if the reason behind the smears is too much target being loaded. I read in the manual that when the blot has a smear like that it can be caused from contamination of the probe with unwanted RNA or DNA. Do you gel isolate the PCR product after the labeling reaction?




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