3
$\begingroup$

I have come up with what I thought was a clever idea: Store the agarose gels I pour, and only cut as many lanes as I need to run later, minimizing wasted agarose (and wasted effort/time) when I need to make many electrophereses using a small number of lanes.

I realized that over time, the EtBr would diffuse out of my gel into the buffer that the gels are kept in. Since I add 2 µl of EtBr to 50 ml gel, and the gels were kept in 400 ml TAE buffer, I added 16 µl EtBr to the buffer, reasoning that now concentrations are equal and no net diffusion will take place.

However, after several weeks, I noticed that the EtBr signal from the gel is severely weakened. What can I do to fix this? I can see the following options:

  • Use higher concentration of EtBr in the buffer (how much?)
  • Change buffer weekly, adding fresh EtBr every time
  • Cover the transparent container with aluminum foil to prevent hypothetical photobleaching

Which one is most likely to solve my problem? Should I just give up, and throw away gels that are older than a day or two (I want to avoid this option, because of the record keeping needed to track when the gel was last made)?

$\endgroup$
  • 1
    $\begingroup$ What I've seen is to keep a bottle of agarose gel, then heat it up to melt, pour what you need into a gel caster, add your ethidium. This way your large gel stock doesn't get contaminated with ethidium and you don't have to worry about loss of ethidium signal. $\endgroup$ – user137 Dec 9 '14 at 0:20
  • 1
    $\begingroup$ @user137 I am saving cast gels in order to avoid the hassle of melting and casting a gel everytime I want to run some samples. The bottle of agarose only saves me the work of weighing the agarose, which is quite insignificant in comparison. $\endgroup$ – Superbest Dec 9 '14 at 0:22
  • $\begingroup$ did you see a reduced signal using the same DNA conc or construct after running it several weeks later in a gel containing no previously run DNA band? I think EtBr can photo bleach so just use fresh buffer with fresh EtBr. Even with a stock EtBr you are meant to wrap it around foil! Old gels are generally not good since they can become quite hard and fragile in my experience so using fresh gels is always best. $\endgroup$ – Bez Dec 9 '14 at 0:29
  • $\begingroup$ @Superbest You could store pre-cast gels without ethidium and then post-stain, but you'd end up with more ethidium waste to worry about. $\endgroup$ – user137 Dec 9 '14 at 0:30
  • 1
    $\begingroup$ Come on @Superbest it takes an insignificant amount of time to weigh and pour gels. Pour it before you settle down to check mails or facebook :P $\endgroup$ – WYSIWYG Dec 9 '14 at 6:23
2
$\begingroup$

To increase storage life: after gel solidifies, dampen it with running buffer. Wrap the gel in polyvinyl chloride. Place in plastic container with a lid. Store in fridge in dark. It will last for a year as long as you re-dampen it with buffer each time you access it. Don't keep it submerged in buffer as the etbr will diffuse out. this is probably the cause of your problem. Notice when you order precast gels (either agarose or acrylamide) they are sealed and only a small vol of buffer is added to keep it hydrated. Additionally, the etbr and EDTA in the buffer helps prevent most mold growth although I have seen black mold grow a few times at around a year.

You can always spike the buffer with etbr adding it directly to the electrophoresis chamber before you run the gel (etbr doesn't even need to even be in the gel for it to work) although I find you get a little more sensitivity with the etbr in the gel. If you spike it be sure to mix and let it sit for about 20 min for the etbr to diffuse.

If you are trying to cut down on cost you are probably using a higher grade agarose than you really need for the application your doing so I would take a look at that

If you end up needing to store gels for rna work, add plain old Clorox bleach to a final concentration of 1% to your TAE gel just after microwaving. It sounds crazy but it works extremely well. You will completely inhibit RNAse and your rna bands will look nice!!!. The bleach also helps with storage of the gel.

$\endgroup$
  • $\begingroup$ OP has already stated that the concentrations of EtBr inside and outside the gel are identical. $\endgroup$ – March Ho Dec 20 '14 at 12:41
  • $\begingroup$ @MarchHo To be sure, just because they are identical doesn't mean there will be no net diffusion. $\endgroup$ – Superbest Dec 20 '14 at 21:07
  • $\begingroup$ I can confirm storage of the bleach gel as well - not only that, but despite not taking care to keep the bleach gel away from RNAse contaminants (keeping in the same box as normal gels, dirty electrophoresis tank) my RNA ran and stained just fine. $\endgroup$ – Superbest Dec 20 '14 at 21:10
2
$\begingroup$

I think I have some evidence that the key factor is light.

Since asking this question, I changed the buffer for fresh TAE-EtBr (same concentration as in my gels) moved my gels from a well-lit area into a closed, opaque box so that they remain in darkness. After 1 week, I ran equal amounts of my ladder in the stored gel, as well as a fresh gel. The results (old gel on left, new gel on right) show very little difference in signal - I conclude that blocking the light is sufficient.

$\endgroup$
0
$\begingroup$

I'm not quite sure what you are doing. Are you running the whole gel each time and cutting off the lanes you've used, or only putting the lanes you desire into the tank and leaving the rest outside?

If the former then the ethidium bromide will be leaching out of the gel because it is positively charged so each time you apply a current to it some of it leaves.

If the latter, I'm not sure why you are storing your gel in buffer at all. Wrap it in clingfilm to stop it drying out and store it away from UV light to avoid the ethidium degrading.

Frankly, however, I'm not sure why you think this will be a big timesaver? You can store pre-melted agarose with ethidium bromide added for a period of weeks and pouring a fresh gel doesn't take a significant amount of time.

$\endgroup$
  • $\begingroup$ It's the latter. I have a big gel stored in a container (easier to open and close than film), and I cut off lanes I need and run only those lanes. $\endgroup$ – Superbest Dec 9 '14 at 17:01

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.