DNA Gel - (Sep/20/2008 )
I'm getting a problem that my DNA gel is too bright (white in color, not transparent) in the photo with short exposure time (0.1 sec), even the marker bands and product bands are shown. My supervisor doesn't satisfy the result, he wants a transparent gel in photo and the result is sharp. I used pre-stained 50ml 1% gel with 2ul EtBr for 300-400bp product bands, I also tried post-stained method in working EtBr solution for 1 hr. The electrophoresis condition is 130V and 10-15 min. Anything wrong? Thanks!
Could you post us a picture?
When you use 2ul EtBr I assume you are using stock concentration EtBr. What concentration of EtBr are you using? When you stain you gel, after running it, what is your working EtBr concentration? 1hr does seem very long. Depending on concentration of EtBr the staining time is between 10min to 20min.
Have you tried destaining your gel in water for 10min to 20 min?
Thanks perneseblue for your reply.
I don't have the photo right now. yup...it is stock concentration (500ug/ml). The concentration of working solution is 0.5 ug/ml. I didn't try destain.
You mean... I can try destain?
yes, you can destain the agarose gel. You can reduce your staining time to around 10 minutes (depending on the thickness of the gel)
EtBr binds more strongly to the DNA than to the agarose. So soaking an EtBr stained gel in water will lower the background fluorescence.
It sounds like the gel documentation machine´s problem or the gel is too thick? Maybe u can use lower exposure time?
But my colleagues are using the same machine, their gel photos are very good. I have already used 0.1 sec for exposure time. Maybe my gel is thick.
Is it also a mistake that I set the gel for long time (around 2 hrs) before running electrophoresis?
as long as you keep the gel hydrated (covered by the running buffer) you can keep your gel for days before loading and running it. Although it would be better if you kept the gel in the fridge, covered by running buffer if you are going to keep the gel for more than a day.
sounds like you have too much etbr, i do it at 2ul stock concentratin/200ml 1% and works every time, even that sometimes is too bright.