Gel electrophoresis trouble

[hsip_15]

Dear all,

In my first trial with electrophoresis, I used TAE buffer as recommended in Maniatis, 2001 which consisted of 40mM Tris-Acetate, pH 7.0 and 0.5M EDTA pH8.0. This trial I failed to obtain any bands including the DNA ladder (Fermentas).

The next few trials, I tried using TE buffer as recommended by Kado and Liu based on the literature by Mickel, Arena and Bauer (Physical properties and gel electrophoresis behaviour of R12-derived plasmid DNAs, Nucleic Acids Research) which consisted of 0.04M Tris-Acetate, and 2mM EDTA, pH 8.1. The latest trial, I had faint bands for my samples of plasmids and stronger bands for the control (pUC19), however, the ladder did not show at all although I have loaded the ladder in wells at both ends of the gel. Why is it so? What could have been wrong?

Another question is, under UV-illumination, half of the gel (the negative end with sample wells) seems to be brighter than the other half. There is a clear difference between both ends somewhere in the middle of the gel. Why is it so?

Thanks alot.

[kmunson779]

The first thing I'd do would be to check your staining protocol. Are you using Ethidium Bromide or something else? Are you adding your stain to the gel, to the samples, or staining post-run?

If using EtBr and adding it to the gel, you'll see that half-stained half-not gel pattern because the EtBr migrates towards the cathode (the end of the gel where your sample wells are). You can fix this by staining post-run or by destaining the gel (washing in water or buffer on an agitator for 5-10 minutes after the run).

If this is not the case, you should check the bulbs on your UV-transilluminator to see if one is burnt out. This will cause dark regions on the transilluminator.

Are you visualizing by eye or using a camera of some sort (polaroid or CCD)? You need a lot more DNA to visualize by eye than with a camera. Perhaps your sample has more DNA than your ladder, so you can see the sample and not the ladder?

I'm surprised that you don't have anyone in your lab who has performed electrophoresis before that can help you troubleshoot. Someone who can watch exactly what you're doing is a tremendous help in figuring out where you're going wrong.

[hsip_15]

Dear Kmunson779,

Thanks again for your prompt reply.

First and foremost, I added EtBr to my gels. So, your answer is of great help to me.

Then, I visulized the gel using both, camera and also by eye under the UV-transilluminator. Both showed the same results.

I have tried asking a few who are doing genetics and microbiology currently and it seems that they are unsure of the reasons.

Thanks a million.

[kmunson779]

If you have access to a camera, I'd suggest trying a much longer exposure time to see if you can see anything at all on your gel.

If you have a high fluorescent background, try destaining the gel so you can see your bands better.

Try a brand-new aliquot of ladder -- this should be a great positive control because you'll know exactly how much DNA you're adding, you know it's clean and what it should look like. If you're using an older tube, you could have DNAse contamination or something else mucking up the sample.