The quality of our DNA electrophoresis (for genotyping) varies from experiment to experiment, and I am trying to understand why. Sometimes bands look crisp and sharp, and other times they have a smearing, comet-like effect. The data is ultimately interpretable, but the poor quality bothers me. We are using the same apparatus (15cm between electrodes), the same buffer (1x TAE), same gel concentration (1% agarose), the same running parameters (100V, 45 min), the same ladder, same loading buffer. The PCR products are made in the same way, using the same reagents, same starting material (~3mm of tail tip). I thought maybe it was running too fast, so I tried reducing the voltage to 75V, but this made no difference. Would anyone have any suggestions for me?
Hi Gisella,
the first think I see is that you have primer dimers which give avery stron signal. This can interfere with the efficiency of your PCR - so if possible I would try to reduce your primer dimers (Hot start PCR, Annealing temperature, Mg-Chloride).
Regarding your weird looking gel electrophoresis I would say the first gel looks good (ecxept from the low running primer dimers).
Regarding the other gels I would think that the gels where to thick and that your gels schould be thinner.
Sometimes there might be also to much from the PCR-Product on the gel especially on gel 2 and 3) - you might have loaded the same volume on the gel, however if the PCR was very efficient you might end up with to much DNA on the gel.
What we did to get better results is that we didn't use a gel chamber but a simple glass plate. We put the comb at one end of the glass plate (we used some clamps to hold the comp in the correct position) and poured arount 15-20 ml of gel (for a small gel) slowly on the glass plate. These gels are very thin and you can only load around 5 µl in the well, however these gels give usually sharp bands.
However if you don't like to use the glass plates you should simple use less agarose in the electrophoresis chamber and use less of your PCR product for the gel run - this should give you already better result.
What might also influence the look of your bands is your voltage and running time - sometimes its better to reduce the voltage and to have a longer run time.
I hope this helps
Dörthe Andrea Kesper Thank you so much for your detailed response. I will try out your suggestions today. Most appreciated!!!
In my opinion, perhaps the thickness of the gel is not even. Maybe you could try casting the gel using a circular bubble level first to ensure the platform is level.
Ahmad Hafiz Murtadha Thank you, Ahmad. I will give that a try. Thank you again!
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