While doing agarose gel electrophoresis, the bands are either very light or not visible despite the amount of good DNA quality and quantity. I am using 1.5ul EtBr for 30ml of gel. I have already ruled out many factors like TBE, agarose, gel running conditions etc. Kindly give some suggestions for troubleshooting this problem.
how do you expose the gel to uv-light? Do you remove the gel-tray before you try to have a look on the gel?. Sometimes it is as simple as that the gel tray is not uv-permeable and therefor the EtBr is not excited.
As you are saying that you use good quality and quantity of DNA. How did you say that that the DNA is of good quality and concentration? If you are saying this on the basis of nanodrop reading then you should know that sometimes nanodrop shows the high concentration but it works in the principle of light absorption and whatever is there in your DNA sample including RNA or proteins all together can show you the good concentration..so you should note down the 260/280 ratio while taking the concentration of your DNA..2nd problem might be with your quantity of EtBr try to take very less amount..if you take more Etbr then your background of the gel would be to highly contrasted with EtBr so you would not able to see your DNA band or it can looks very faint...so sort out these things..
hope you got my points..
good luck.
Are the bands of your size marker clearly visible? If so, then it is likely that your PCR is not working.
I suggest make your gel very thin, i causes your band get sharper, and concentration of etbr is important(EtBr stock solution should be10mg/ml) and you should add 5ul of EtBr stock to 100ml gel. as Andereas says,check bands of your lader,are they clearly visible?!
good luck
Also, another question is the size of the fragment. This falls in line with what Andreas was hinting at. EtBr migrates across a gel in the reverse direction of the DNA. If you run your gel for a long time, you might be visualizing a part of the gel that contains little, if any, EtBr. This could potentially be solved by inundating the gel in an "EtBr bath".
Secondly, I have rarely heard of people going below the approximately 1/10000-fold dilution for EtBr. That would mean 3 uL of EtBr for a volume of 30 mL. Since I've never tried, I don't know how it affects the gel overall.
What would be the purpose of the electrophoresis run? are you using it to evaluate DNA integrity, i.e. in a 0.8% agarose gel? or is it intended to evaluate a PCR reaction? Please, be more specific, for it is not clear if you are asking about your gel separation or the PCR yield. If you are running gDNA you yould use a proper marker (i.e. lambda DNA) to check for the size of your fragments and also for the quality of your gel, If the question is about PCR, why do you imply that the problem is in your agarose gel?
In addition of previous comments that were given, have you checked the camera setting? :)
You should try staining gel afterwards instead of adding EtBr to the gel,,,or make sure add Etbr just before pouring the gel,,,do not boil the etbr.
during electrophoresis heat up problem may be there. Electophores in fron of AC or fan
- Badges
- Science method