I have been trying to resolve ss DNA with varrying nucleotide length, ranging between 20nt---86nt long in a 20% and 15% denaturing PAGE with urea of 8M. However, my bands are not sharp enough and disticnt bands were not observed. The DNA ladder used (10 bp) did not appear sharp and distinct either. I used 6x orange loading dye and I denatured the samples and the ladder at 95 degrees celsius for 3 minutes and kept them on ice immediately prior to loading. I run the gel with 1X TBE at 60 volts for 3 hours and stained with 0.8 X sybr gold. Also, I realised the samples do not fully sit in the well. Please, is there something I am not doing right? I have attached the image for clarification.
Dear Dorothy, I think your gel may not be in good condition. So, check your reagents to make sure they are not expired, and in particular APS solutions should be made fresh and used within a couple weeks when stored at 4C degrees. You don't want to store this reagent at -20C degrees for long period of time. Another thing to do is to de-gas your gel solution mix before adding in APS/TEMED and pouring the gel. Finally, dissolve your samples in denaturing loading buffer containing formamide. Following just a recipe from ThermoFisher: A 1–2X solution of 95% Formamide, 18 mM EDTA, and 0.025% SDS, Xylene Cyanol, and Bromophenol Blue. Supplied in one 1.4 mL tube. All Ambion® Gel Loading Solutions are rigorously tested for non-specific endonuclease activity, exonuclease activity, RNase activity, and for functionality. Hope this would help.
Label these with isotope and run the old sequencing gel. or label these with fluorophore and run on capillary sequencer as ABI310...
perhaps the gel was too thick, not enough material loaded or something, If she has a capillary would be the best solution
If you are certain that your gel components are good, you may wish to consider a loading buffer that contains formamide to assist the denaturation. Then load the samples while hot. Partial renaturation of your samples when loaded can result in fuzzy bands.
We used to do this a lot in the old days to purify oligos by UV shadowing (rather than using any label). I recommend you follow Theresa's suggestion above and denature your oligos in a formamide loading dye before applying on the gel.
I also wonder how you calculate 8M for urea in the gel mix. 6M is about the max I could ever get.
Finally it also helps to deionise your gel solution with amberlite or any other mixed bed resin before use.
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