I have been running DIG Southerns. I have no trouble getting single bands to show up at sizes of 8 kb or smaller, but bands at high molecular weight (15 kb or higher) are less distinct, often showing up as smears with the expected band at the top of the smear. This would be less of a problem except that the band is not distinct enough from the smear to identify clearly. The probe is ~700 bp long; we digest the DNA overnight and run it several hours at 50V on 0.7% agarose. Does anyone have an idea how to make the high-molecular weight bands more distinct or cut back on the smear?
I encountered similar problems when my DNA was not completely resuspended. Basically there was quite a lot of partially dissolved DNA in the upper region of the gel (and subsequently the blot). In this region the probe could bind and this led to smears, dark patches, etc.). Maybe you have a comparable problem?
Christians is a good suggestion and may I add that the gel constitution looks good but I do not think it has run for long enough. It depends on the gel size but with 20cmx20cm gels running overnight at 3-5 vols/cm worked well ans running in a cold room minimise thermal diffusion of the bands. Using a double digest might make the larger bands smaller then you would not have an issue with separating large bands. Also as it is a large band if you are transferring by diffusion on to the membrane it sometimes makes the transfer more efficient if you partially degrade the large band by masking the small band area of the gel and leaving the large dna fragment area exposed to the transilluminator for a minute extra. The dna breaks so transfers better but is still in the correct place for a 15kb band of course
I appreciate the responses. I usually run the gel for these at 35V but my PI suggested 50V as potentially resolving the smears, which unfortunately it did not. We run smaller gels for Southerns so we cannot run them out overnight. I have had cases where the large bands resolved acceptably, but it is not consistent. My suspicion is that Dr. Scheckhuber is probably correct. After digesting, the DNAs (10 ug each) are ethanol precipitated, dried in a speedvac, and resuspended in 25 uL TE (pH 8). Perhaps I am overdrying the pellets or not using enough TE, making the resuspension difficult.
Try to use 100 µl TE instead of only 25 µl. Your final concentration is still high enough for most applications. Don't load too much DNA. 500-700 ng of genomic DNA per lane is usually OK for small gels. If you have plasmid DNA you should use much less DNA, of course (10 -20 ng / lane). Good luck!
since the separation is not reproducable perhaps it could be a salt problem with some samples having too much salt after the precipitation and loading slower than low salt samples as the current would be lower in the high salt lanes. Possibly an extra 70%ethanol wash is a thought or resuspending in half strength TE
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