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
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2$\begingroup$ That is a lot of EtBr. You are using about 30x more than what most people do! You're potentially exposing yourself to a higher amount of a dangerous chemical than usual. $\endgroup$– SuperbestCommented Apr 1, 2014 at 22:58
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$\begingroup$ What's the typical amount? My graduate TA told me to run 10uL per 25mL for the gel. $\endgroup$– kwiromeoCommented Apr 1, 2014 at 23:10
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$\begingroup$ Standard amounts are about 16 ul per 100 ml of gel IF added in the gel solution. $\endgroup$– user560Commented Apr 2, 2014 at 1:43
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1$\begingroup$ EtBr is a powder that can be made to solutions of different concentrations. So volumes don't make much sense unless the stock concentration is mentioned. Final concentration of EtBr in the gel should be 0.5$\mu g/ml$ $\endgroup$– WYSIWYGCommented Apr 2, 2014 at 4:42
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1$\begingroup$ @Chris Yes. However, I've heard a nice analogy - "it's kind of like smoking a cigarette". I, personally, don't smoke. $\endgroup$– SuperbestCommented Apr 2, 2014 at 5:57
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1 Answer
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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.
answered Apr 1, 2014 at 23:14
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$\begingroup$ The blue LED transillumination is the problem. There is nothing wrong with making your gel, electrophoresis or resolution of bands based on your image. EtBr is best illuminated in the short UV range and is a function of wavelength. It's worth comparing abaorbance ratio for peak UV illumination vs the wavelength of blue LED (415 nm? ). 20 sec exposure is awfully long. What do 100 ms to 1 sec exposures look like? $\endgroup$– user560Commented Apr 2, 2014 at 1:47
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1$\begingroup$ Since this answer was accepted, let me clarify: Looks like the problem is that the wrong illuminator was used. The linked device produces blue light, for use with CYBR Green. For EtBr, you need UV. Probably the signal you did get was from UV bleeding from the blue LEDs of the device. $\endgroup$ Commented Apr 3, 2014 at 2:49