Always when I run agarose-gels I get these droopy bands from my DNA ladder (and sometimes samples), and based on the sample bands I get in different experiments, the true sizes in the ladder seems to be higher than the actual ladder bands. What causes this? I have run 1%, 1.7% and 3% gels, at 150, 100 and 50 V, with 100 bp (100bp-1000bp) and low range (25bp-700bp) DNA ladders, I reuse the bottle but make sure to remove all old agarose before mixing new agarose. I always wash everything and add new TBE buffer before running my gels, and I have used both diluted DNA ladders (with extra loading dye) and undiluted DNA ladders with volumes according to manufacturers protocol. They seem to even out at lower sizes. What causes this? Will this problem go away if I use new bottles every time, or are there other things I can tweak?
This could either be because of some fault in your gel running buffer. From your question, it's not clear whether the bands in all the wells are DNA ladders or some of your samples. If they are your samples then it's the buffer that has to be blamed. But if you observe this only for your DNA ladders alone then it is caused by the degradation of the ladder DNA due to improper freezing and thawing. I suggest you use a fresh batch of ladder DNA to verify the same. Hope this helps.
Hello Amanda, how are you? So, several things can cause this problem. This smiley bands usually appears when you run your gel in high voltages, when you performed your PCR with high concentration or large volume of DNA sample or DNA ladder, or the agarose was not well prepared. In the photo the bands do not appear straight with the ladder, for me it is a problem with the gel, the agarose was not well dissolved when you prepared. The arrested bands look like you performed your PCR with a high concentrated DNA sample and the remaining DNA will arrest when you run your PCR product. Be careful when you change the ladder or the loading buffer volume and concentration. High values of the loading buffer or if you adding the DNA revealer (SYBR safe, Ethidium Bromide, or other dye) when you preparing the gel, could also affect the DNA running, especially in bigger fragments. I generally run my PCR products in 1% agarose gel in a 0.5x TBE at 100V during 40 minutes, I am not work with more of 1000bp fragments for DNA and 300 for cDNA. If you working with small fragments make sure that agarose is well dissolved when you prepared 3% gels. Good luck.
Hello Amanda Westergren Jakobsson
As you said, the smiley bands come not only in DNA ladder but in samples also, I think you should check the buffer quality and concentration. Ionic potential and pH of TAE/TBE buffer are crucial for the proper DNA sample migration in agarose gel electrophoresis. Check the buffer quality or prepare fresh buffer and sterilize it before using. Before pouring the warm agarose solution into the tray, make it a homogeneous solution, otherwise the agarose gel quality may be affected.
Best wishes.
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