When you're setting up your gel, make sure to use a comb that's in undamaged shape (no nicks or deformities) so you can create wells that have smooth and straight edges. Before you load the ladder, take the time to mix it thoroughly with the right loading dye; otherwise, you might end up with uneven separation.
Be mindful of the amount of ladder you're loading into the wells. Too little and you'll have a challenging time seeing it on the gel; too much and you risk smearing, which can make interpretation difficult.
Make sure that the gel percentage is appropriate to the size that you are interested in. Small dna higher agarose concentration. Make sure that the gel is completely set and has cooled to room temperature before running the gel.
Make sure that the loading buffer/dye is well mixed before loading . Do not worry if the whole lader is not well separated.So long as the band sizes are clear in the region of dna size that you need the rest of the ladder is of less importance, A picture of the gel would help to see the problem
That's quite a long run time. Typically, gels are run at a higher voltage for less time. Is your product size unusual?
Is your gel getting warm/hot by the time the run is finished? If you gel is very warm/hot the bands can get fuzzy.
I would also suggest using fresh running buffer for each gel. Dump it out after each use and rinse your running rig with water and let it dry.
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