Hello all,
I have been running sequencing gels for quite some time now and I find that the bands start broadening as the dye reaches the halfway mark (pic attached). I followed the publications in the Electrophoresis journal from the 90s and they seem to mention the current to be the problem, but I have run the gel at 7W, 20W, and 40W while getting the same result. My setup is a 40 cm 0.4mm denaturing PAGE with urea. I also checked the heat distribution with a laser thermometer and the temperature is uniformly ~43C across the gel.
I'll be grateful if anyone can give me suggestions to prevent this problem.
Best regards,
Subramaniyam
How do you prepare your buffers?
Do you use too much acid or base for reaching right pH?
I make 10X TBE first with 0.89M Tris HCl pH 8.0 (diluted from 1M), 0.89M boric acid, and 20mM EDTA.
Most important cause of high temperature is too much of free ions in buffer
Check this
Actually the generally recommended gel temperature is around 50C (theoretically), so I don't think that temperature might be an issue. To check this I also used different wattage, and the heat generated across the gel increases with higher number of watts, but the end result is the same in all conditions.
Subramaniyam Ravichandran
Sorry it was my mistake. I did not attention that you mentioned denatured gel.
is band broadening really a problem to you.?
The dye markers often used with dna (bromophenol blue ,xylene cyanol and orange G ) are all small molecules and will be expected to thermally diffuse much quicker than dna or protein so I am thinking that your sample bands may still be clean and tight even though the marker dye is spreading. The temperature of the gel between the glass plates may be much hotter than the plates themselves which are losing heat to the environment so your gel temperature could be much hotter than the 43c measured and this would be correct
I wonder if I misunderstood the question and it is the second blue band that you are interested in. This is probably caused by the pH causing the dye to form a new conformation which may suggest the buffer is becoming depleted and the acidity is increasing. In this case the blue dye may turn yellowish if run much further. If this is the case try replacing the running buffer part way through the run . If this makes a difference then you have buffer depletion and your running buffer is made up wrong so prepare new running buffer taking particular care with the dilution of the buffer concentrate mentioned in your answer above
Thank you very much for your reply. My question was not pertaining to the dye broadening, though. I am attaching the developed autoradiograph of my sequencing gel for A/G with formic acid for your perusal. As you can see, the bands are clearly sharper on the upper half than on the lower half. I am running a 20% urea PAGE gel of 0.4mm thickness in 1% TBE of pH 8.0.
Best regards,
Subramaniyam
OK ignore what I said earlier . Fuzziness in sequencing gels is often caused by the temperature being too high or by denaturation not being complete. I do not know the formic acid technique but formamide up to 40% as well as urea is a good idea but the formamide must be de ionised fresh before use or acidic products build up and resolution becomes poor.Check also that the acrylamide is good quality or again acid residues can accumulate. You can get a similar effect with glycerol interacting with the borate in TBE but you are probably not using glycerol to weigh down your samples in the loading dye. If all else fails borrow acrylamide/bis/ temed from another researcher and make up new APS in case reagents are getting old
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