I have a recipe of loading dye (10 mM EDTA, 1X TBE, pinch of Xylene cynol and Orange G dye which is made upto 10 ml with 100% Formamide) but somehow the bands of the nucleotide product (enzymatically degraded DNA) are not distinct and well defined or fuzzy. I use 1 X TBE buffer (freshly prepared), the volt used to run the gel is 1500-2000 Volt or 25-40 W. Could you please suggest the recipe of loading dye or other factors that could give crisp bands for publication purpose. Any advice would be highly appreciated.
Thank you in advance
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You could try heating the samples in the formamide loading buffer to 90c for 30 seconds before loading and make sure that te wells of the gel are syringed with running buffer just before loading. If you are running alternate wells then load the empty wells with loading buffer (diluted) so that all of the wells have the same amount of salts. If this fails then try deionising the formamide or using a newer batch of formamide
Thank you for this valuable suggestion. How can I deionize the Formamide?
You can stir 1g of mixed bed resin ( Sigma M8032 or equivalent) in 10ml of formamide for 1hour then filter and store aliquots or I used to just leave the resin to settle in the bottle and take off the supernatant for use. The main contaminant is formic acid which may lead to depurination of dna so it is worth buying good quality formamide or deionising it . If your institute runs its own sequencing and genotyping then they will have top quality deionised formamide and you could borrow a little just to test if it makes a difference
Sequencing gel migration is very critical to what are you loading, especially if your DNA fragments are relatively long >100 nt. If you have some proteins, I recommend phenlysing and EtOH-glycogen precipitation of the samples prior gel loading. Urea gels do not like salts, if NaCl is > 40 mM precipitate the sample before resuspending in the gel loading buffer. Also, after a couple of minutes heating at 90°C before loading, I keep the samples in ice during loading.
All enzymes that work on DNA have Mg2+ as a cofactor. So, I think the EDTA you have in your buffer will already do the job, as it complexes the Mg2+. Concerning proteins bound to DNA, an alternative to phenol extraction is to use proteinase K for the deproteinization of the samples.
Thank you so very much to all. I am working on it. Your valuable suggestions are well taken.