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Gel Running Crooked?? Please help..


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#1 ThePipetMan

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Posted 30 May 2010 - 05:15 PM

I use Invitrogen 4-12% bis-tris pre-cast gels, 20x MOPS SDS running buffer, invitrogen gel boxes and biorad power boxes. The other day while running a gel, the dye front was migrating fine, until suddenly, in the middle of the gel, one side started running faster than the other, and when I came back to check on it (literally within 10 minutes) it was VERY crooked -- i.e. one side had migrated so that it was an inch or so further down than the other one.

This happened on BOTH gels (I was running two gels in the same box). I stopped the run, tried to adjust the position of the gels in the box, and refilled with buffer, and then completed the run. It "caught up" a little bit, but the results after antibody were awful, as several of the lanes had "blended" into one another and ruined the film.

The other gel box (2 more gels) that I was running -- with the SAME power supply -- were FINE.

My question is: WHAT CAUSES THIS? And what can I do to prevent it from happening in the future? My buffer levels were high enough...it was covering the gel. There did not seem to be any problems with the gel itself.

The procedure I used was identical to the one I've always used, and I've probably done 300-400 Westerns and this has never happened before. Briefly, I run at 100V stacking, then 150V for the rest of the gel, with the gel boxes at room temperature but in an ice bucket w/ice. I use 4x LDS sample buffer, 5% DTT, 10 minute boiling beforehand. I am thinking that it is not a sample problem, but maybe something disturbing the electrical field. Yet...HOW can this be fixed?

Thanks so much for any help you may have...this is incredibly frustrating.

#2 HomeBrew

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Posted 30 May 2010 - 06:23 PM

No problem with the power supply, buffer, gels, gel positioning, or other boxes probably means that one box or its lid is damaged. Did you check the wires? The poles? The leads?

#3 Prep!

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Posted 30 May 2010 - 08:25 PM

No problem with the power supply, buffer, gels, gel positioning, or other boxes probably means that one box or its lid is damaged. Did you check the wires? The poles? The leads?



i second tat.. there may be some problem with the platinum wires tat completes the circuit at one side... it might have been damaged.. may be during washing or cleaning and u might not have noticed it.. why dont u run a different electrophoresis unit if u have and u can figure out wat s wrong with the unit!!!
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#4 ThePipetMan

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Posted 31 May 2010 - 07:40 PM

The boxes seem fine: but I have a theory:

If the box were tilted slightly and there isn't enough running buffer in the inner chamber (that covers the wells of the gel), so that only part of the gel is covered, would this cause the uncovered part to stop running and the covered part to keep running?

My theory is this: I run the gel box in an ice bucket, so it's never exactly level horizontally. I think maybe it got bumped around a little bit and some of the running buffer splashed out / got bubbly and didn't cover any more. Since the box was tilted, one side remained covered while the other did not.

Would this cause the bizarre running behavior (that seemed to catch up after I stopped the gels, added more buffer, and repositioned)?

Thanks again for the help.

#5 bob1

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Posted 01 June 2010 - 05:24 PM

The boxes seem fine: but I have a theory:

If the box were tilted slightly and there isn't enough running buffer in the inner chamber (that covers the wells of the gel), so that only part of the gel is covered, would this cause the uncovered part to stop running and the covered part to keep running?

This was going to be my answer, it happens all the time if your gaskets don't seal so well and the upper chamber leaks a little.

#6 laurequillo

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Posted 01 June 2010 - 08:12 PM

To avoid that I fill the chamber with buffer. New buffer inside, used one outside, and completely full. In that way the inner liquid does not goes out!
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#7 ThePipetMan

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Posted 02 June 2010 - 01:48 PM

THANKS so much for the help you all! I'm nearly positive that the problem was a slight leak in the inner chamber, and that combined with the gel box being a little bit tilted to one side meant that only half of the gel was part of the circuit for a good 10 minutes (I had an extra gel that I used to test this by just running 2 ladders, and I replicated the same effect).

I've also been using fresh SDS for the inner chambers / used for outer, and am only using it once. I've run 5 gels since then and all turned out fine (*fingers crossed*) -- so hopefully this issue is resolved.

Again, thanks a lot!! :wacko:

#8 HomeBrew

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Posted 02 June 2010 - 05:24 PM

Put a pedestal (a tip box, perhaps?) in the ice bucket, place your gel box on that, and fill the sides with ice, or run the unit in a walk-in refrigerator. If you just jam the unit into a pre-filled ice bucket, you're bound to get it crooked, or it will become crooked during the run, as the ice melts. Have the unit flat on a stable platform.




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