Agarose gel electrophoresis troubleshooting - (May/01/2009 )
I was wondering if someone could help. The last 3 days or so, my gels have been looking abysmal. The dye almost fades from the gel as it runs, so when I finally image it the bands are so faint that I have to use long (2s) exposure and then tone down the image intensity. Here are some details.
I use a 2% gel for bands that range from 250-600bp. I just switched to a new bottle of agarose, same brand tho. I use TAE buffer that I re-made but still have the problem. I usually make the gel, add EtBr and pour it into a mold. I load 10uL of sample into the wells after adding some 6x loading dye.
The bands, as the gel runs, just seem to fade. The ladder does not image well either, which makes me believe it's not my samples. I have re-made the TAE buffer and am now trying another lab's TBE buffer to see if that will help.
Anyone had a similar issue??
Try adding EtBr to the running buffer as well. If you just add it to the gel, it will sometimes run right out of the gel.
Instead of adding EtBr to the gel, soak the gel in an EtBr solution after the run, then destain in distilled water.
What tracking dye are you using? Most tracking dyes are pH sensitive, so your "fading bands" might indicate a pH problem, which might further indicate a problem with your gel buffer.
You're not accidentally making your molten agarose with water, are you?
No, I'm not using water... I just tried a TBE solution prepared down the hall. They put EtBr in their stock, so I used it to make the gel and to run it in ... and it was still so faint! At the end of running it (I only even ran it for like 30 min) I could barely see the dye in the gel. I used a 2 s exposure and could see only the faintest bands.
And regarding the dye, I used one dye to mix with the samples and another dye to make the ladder... and both were not good.
I didn't check the pH of the TBE buffer. When I made the new TAE yesterday I made sure it was around 7.5 .
I am going to try the other labs' agarose next. It seems it HAS to be the buffer that's the issue - but even with all the changes I've made I don't know what could be wrong! Frustrating.
I will be back in on Monday to make a new gel. I am going to use the other lab's TBE AND their Agarose. Thanks for all the suggestions guys!!!
Always make sure you use fresh buffer everytime you run a gel (or at least when you're running important samples).
If the buffer is reused often, it is not of much good.
I'm just wondering if using SYBR Green can be an alternative method or not...you know, EtBr is quite toxic...
I used SYBR Green and nothing like that happens...
Lazinase on May 8 2009, 02:08 AM said:
etbr may not be as toxic as you think, check the links in this post.
I don't really think that the problem is with the buffer itself, because if you have changed it and you still have the problem, it must be elsewhere. I don't think that the problem should be with EtBr either. I usually put it in the gel, not in the loading buffer.
What I recommend you is to clean the cuvette, the mold and the comb with water and soap very well before using it. You may think this is a stupid thing, but if somebody has used it to run RNA samples, for example, and it has not been cleaned, some rests of formamide, etc, may remain, and this will spoil your gel for DNA.
Of course, check the pH of the running buffer, check that the loading buffer is OK, and so on.
Oh! I was forgetting to tell you that I prefer EtBr rather than Syber Safe because Syber Safe loses its intensity much faster that EtBr (at least from my experience).
You're looking at some pretty small fragments, and EtBr migrates in the opposite direction from DNA during electrophoresis. Because of that, you can get uneven ladder staining and inconsistent staining of fragments. I recommend staining in an EtBr bath after the run, also because this gives you sharper resolution and is better for quantification.
If you're wondering if the buffer is getting spent during the run, check its pH after the run both in front of and behind the gel. That should only really be a problem if you're doing a long run, though. TAE is fine for me up to a few hours, but is exhausted faster than TBE so I'd try TBE for longer runs. I've had to do some fairly long runs before, and for these longer runs the dye will dissipate and fade.
I'd also check the quality of your DNA to make sure you aren't getting protein or nuclease contamination. Salt concentration can also be an issue, so you could try ethanol-precipitation to clean that up.
Good points, ickyostes!
I've never added EtBr to the molten agarose for just those reasons -- I've always (three different labs and 20+ years now) stained/destained my gels after the run, for just the reasons you point out.
Checking the pH of the buffer in the two different chambers after the run is a good idea, too...