I've been trying to a good contrast for my DNA bands, however I haven't been so successful. I've been running the ladders only to make sure I get a good contrast before further experimentation. I created the gel using 70mL 1X TAE buffer, 0.84g of agarose (so about 1.2% gel), and 28ul EtBr. I set the electrophoresis box at 90V for about 1 hour hoping to get a better gel but the results came out looking the same as if I had ran it at 110V. I've attached a picture of my gel. It was taken with a DSLR, no flash, 20 seconds exposure time. Any tips would help. Thanks
I normally use 1-2 ul (depending on whether my gut tells me I have a lot of DNA, or a little that day) in a 70 ml 1% gel (which is probably 60-65 ml after boiling in the microwave). I have no trouble whatsoever seeing ladders and DNA samples of down to 100 ug in wells not much bigger than about 4 mm.
Here are the factors that you can manipulate to alter the brightness:
- Amount of EtBr. More is brighter. (However, you already use a lot - using less may even be better because it would reduce background while still saturating the DNA with EtBr)
- Amount of DNA. More is brighter. To avoid guesswork, use a ladder and consult the instructions in its manual or spec sheet. They usually have an example picture of a gel, and say exactly how they obtained it.
- You messed up. Do other people in the lab get good gels? Ask them to do the protocol with you.
- Sample preparation. Are you using a loading dye in the correct amount? Is the DNA diffusing out of the wells? Are you spilling a lot of it when you load?
- Band width. Obviously if you load the same amount of DNA in a well 4 times as wide, it will be 4 times fainter.
- Strength of UV light. Many illuminators have a knob to adjust the intensity, people turn it down to avoid damaging DNA when gel-purifying restriction fragments. You can also play with intensity, gamma and exposure in a geldoc machine.
- Quality of EtBr. I've never actually had this happen, but your stock may be bad for some reason. Ask another lab for a ul or two to easily control for this.
- Quality of DNA. Obviously, your sample must actually have DNA in it, and not be degraded. You can check amount and purity easily with a spectrophotometer, but it will not tell you if the DNA has been digested into its component nucleotides (that is usually checked with a gel).
- Amount of gel. The crucial variable is obviously not total amount of EtBr, but concentration. Less gel, brighter bands - but you seem to be using a standard gel amount.
- Quality of gel. Again, never had this happen, but try making the gel with someone else's reagents.
I can see that you have a ladder in the middle, so at least some DNA is definitely there.
Also, keep in mind that off-the-shelf cameras are not good at imaging the EtBr fluorescence, because the wavelength is a bit unusual. The nice pictures you see in papers are taken with a specialized geldoc machine or a camera with an appropriate filter.