Nupage Bis/Tris Current Voltage problem - Current is so high! (Mar/22/2010 )

Basically, I connected everything according to Invitrogen's protocol fo the 4-12% Nupage Bis/Tris gel with MES running buffer. When I set the voltage at constant 200V like requested, I should see 100-140mA, but I see 400!!!!

Question I have is : What effect does high mA have on the gel? Does it cause heating and ruin it causing a bad run?

Also, does the same buffer have to be used for both inner and outer wells? Because I used a newly made up solution for the inner well, cause I ran out of solution when making for the outer well. Is this why the currents are all messed up?

Also my dye front is running curved, like a frown, intead of a straight line. Is this caused by the high currents, i.e it has not stacked properly?

Also, does anyone have any experience with working with expired gels?

Sharky on Mar 22 2010, 09:10 AM said:

I've worked with expired Bio-Rad gels. They ran terribly. I've read another thread here about expired gels, and many say they haven't had problems. The one time I tried, I did. I think expired gels could cause the problem you're seeing, if that's what you were using. The new buffer shouldn't matter, unless you used a completely different type of buffer (MOPS instead of MES). I use the 4-12% nupage gels frequently with MOPS buffer, and haven't had any problems running between 180 and 200 V, but I've never checked the mA to see where it runs. Are you using commercial MES buffer or are you making your own? If your own, is there any chance of an error in formulation?

Cheers for the response Fish doc. Expired gels may be the culprit, like you state. Invitrogen reckons it could also be a high salt content in my samples, but I used Bugbuster reagent which is compatible with electrophoresis. I am making commercial MES buffer, so it should be fine. Overloading can also cause this frown shape apparently. I am going to see how good the gel looks after picture. I am not really worried about molecular weight for now, I jsut want to see big fat overexpression band!

Cheers

If the buffer in the inner buffer overflows and touches the outer buffer can that cause current spikes?

When I run my invitrogen Bis-Tris 4%-12 PAGE 3.5" gels with vendor made and homemade MES at 200V, my mA is runs at 400 consistently with several different brands of power supplies. I don't think this is the problem.

I wouldn't lean towards overloaded gels to run like a smiley face, they are just messy and smeary. Buffers are more often the problem. Either the buffers that your sample is in, or using old buffers in the gel rig.
Some samples have to be dialyzed into a more compatible buffer before being loaded in the gel. Check your handy dandy invitrogen pamphlet for compatible chemicals and concentrations.
If the chambers (inner and outer) are not sealed off from each other properly, the power unit won't run. It messes up the circuit. (I'm not an electrician for sure)
If you're buffer is overfilled at the top and the inner and outer chamber buffers are in contact with each other, the same happens.
If one or both of the champers aren't filled to the top, (without flowing into each other), you can get a smiley face pattern or other funky things.

You should use the same buffer in both chambers, and of the same age or number of uses. I use my buffer 5 times max to get nice pretty gels every time.

Here's a link to the "Hall of Shame" of bad gels with pictures and the explanations.

http://www.ruf.rice.edu/~bioslabs/studies/...e/sdsgoofs.html

http://www.ruf.rice.edu/~bioslabs/studies/...goofs/misc.html

Cheers for the Hall of Shame links danny. I will take a look at them. I have a feeling my curved running is because of buffer then. I realised I use just distilled water for making my buffer. Not sure if it is deionized, maybe that is causing the high current. I will try using deionized, distlled water 2morrow

Ahir

Sounds like a good plan
The Hall of Shame site is a riot!

Denny on Mar 25 2010, 04:08 PM said:

I dont believe it Denny! I used deonized, distilled water, made sure I did not overload, ascertained that Bugbuster reagent in the sample is not high salt content and is suitable for electrophoresis, and I am stil getting this high current problem and consequent frowing effect.

I noticed that unless the outer and inner chamber MES buffer touch, the voltage is 200, but the current is like 8-10 and very few bubbles can be seen. If its very low to 0 ma it means the process aint working right?

Sharky on Mar 30 2010, 08:51 AM said:

Do you use the Xcell SureLock cells for electrophoresis? I am wondering, because if you do I'm not sure what you mean by the outer and inner chambers touching. How much buffer are you putting into the inner and outer chambers?

fishdoc on Mar 30 2010, 07:20 AM said:

Hey Fish, I dont use the Xcell SureLock. I use the Cleaver Scientific CVS10D Electrophoresis apparatus. I put enough inner buffer to cover the wells, and the remainder in the outer. Its about 200ml / 800 ml respectively. The weird thing is, unless I put enough on the outside to make the buffer connect, I get pretty much no current(around 16ma)