First of all excuse the bad english, I have been doing electrophoresis in agarose gel to visualize PCR products, however I've had problems getting a clear straight image of the bands, since they appear as distorted bands (W shaped bands or wavy bands) (Also frowny bands). I already tried to modifying the voltage (60, 70, 75, 80 and 85 V) I also tried to modify the concentration of agarose (0.8% and 1%) Each time I prepare new agarose and TAE buffer, I also changed the instrument and got the same results. I varied the amount of sample that I placed in the wells (1,2,3,4, 5, 7, 10 and 12 microliters) as well as the sample-loading buffer ratio. The molecular weight marker (always at the ends) is also affected, even after using a different marker. Any suggestions? The fragments that I hope to obtain are of 600 and 2000 bp approximately. Distance between electrodes is 12.5 cm. I´ve also tried to mantain a cold environment for the elctrophoresis.
Try to use a new agarose (not the one that you are using). If you still have the same problem, try to use a different instrument. Good luck
I already change the instrument, but i´ll try with different agarose, thank you.
I think the problem should be with agarose gel or the gel was incompletely immersed in electrophoresis buffer. If you need more information, you can follow the guide: Troubleshooting Guide for DNA Electrophoresis.
Seems like there is a problem in agarose gel. Even the ladder is not clear. Try to make new gel and definitely it will help you to visualize your PCR products. good luck!!
How long do you run it for?? You might not be running it long enough which is why your getting the smiling bands. I tend to run my gel at 90V for an hour and 40 minutes and that works best for me. A good indicator is when you sample is running how far down the gel has it traveled before stopping it. Hope this helps.
Hello Carlos,
Looks to me that you are having a problem with the gel. Either is agarose not ok or during the run is not a good run. Actually, you should try to change the batch of agarose and prepare new working solutions or even stock of buffers for both the gel and the running. Is your gel ok as aspect I mean same width all over, non boubles and so on...
Since also the marker looks bad is for sure the gel. I do not think that you should change many instruments just take one by one the factors I said ...
Generally, is not so difficult to do a gel if you take a bit of care at some essential factors:
1)buffer need to be the same in gel and running cassette, same
batch same conc. 1X generally, can be TAE or TBE;
2) during the run should be constantly the same voltage;
3) the gel should look without boubles, uniform in width and well solidified, you can even put it in 4C if you are in a hurry but please wait till solidifies after you can move it and keep it there for a while;
4) higher temp during the run could influence the quality of the outcome so if very hot run it in cold room or in ice put the whole cassette , depends how long is your run and what are you looking for....
Anyway, in your case I understood is only to check the single fragment from PCR so should not be very very long...
Last the conditions are good in electrophoresis when at least the marker look very well..
Best of luck.
Stefania M. Mang
Hi.
Try to use new gel,and also check the attached files, it might be useful.
Thank you all, what I did was increase the voltage to 90 V, since before I ran the gel to no more than 75 V, and I left it about an hour, I also noticed that it was probably not melting the agarose completely, now I melt it until it is completely transparent. Besides that, I did not change anything. I got straight bands. Thank you very much.
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