Hi, my problem is about EtBr in agarose gel. I tried many times and saw that EtBr is not diffusing well within the gel. It seems that one side of the gel is stained by EtBr but the other is not. So during the imaging, some samples are not visible. The proper staining side changes from experiment to experiment. I tried many times with the same PCR samples and also eliminated other factors such as running buffer, gel preparation, etc. I have never used the running buffer more than once by the way. What can be the problem? I use 1/1000 fold dilution of EtBr and add 2 ul to 3% agarose gel. Thanks!
You should either add EtBr in gel after cooling it to 50 degrees and then mix properly before pouring in the caster. It diffuses well in gel.
If you are facing problem yet, you may try as suggested by Paul. After staining of gel with EtBr buffer. We were doing this when we ran PAGE. And after gel staining u need to wash the get with EtBr free buffer and take the picture.
You need to handle this step as the whole buffer has EtBr.
Try photographing the gel then soaking the gel in 1:1000 etbr for 30 mins and photographing again. If the missing bands re appear then etbr is leaving one side of the gel quicker than the other side and you may want to check very carefully that the gel apparatus is level and the buffer covers the gel to the same depth over the entire gel. Check also that the electrodes in the gel tank are not broken so delivering uneven voltage across the gel width. So long as you swirl the gel/etbr well before pouring the gel then I would expect the distribution within the gel at the start of the run to be uniform.
Ethidium bromide migrates in the electric field, so if you put the stain in the gel before electrophoresis, it will be unevenly distributed afterwards. To get even staining, don't put the EtBr in the gel. Instead, after running the gel, soak it in buffer containing EtBr for a short time. Wash the gel in stain-free buffer briefly before photographing. The staining solution can be reused many times until the EtBr is depleted. Remember that EtBr solutions should be handled with care.
Try to do an after stain as said by paul rutland. You ensure that the etbr is completely mixed up in the solution.and also the tankbuffer is filled properly while running the gel. If all these things are ok and the problem still persist then observe if your dna also shows a similar behavoiur like etbr by doing after staining. If the dna also shows tge same type of movement then check thepowersupply in the electrodes, if it is functioning all aver tge length or a part of it ... Particularly the negatively charged electrode
It is better to add EtBr on gel when the temperature is cool down to about 50 °C (about when you can comfortably keep your hand on the flask) and swirl to mix, taking care not to introduce bubbles.
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