I have a question: why are you loading the samples twice (with a time gap in between)?  I'm looking at the photo but am not sure what you mean. I see a "line" running horizontally through the middle of the gel - I don't know what that is. I also see "shadows" near the bottom of the gel around the sample, and in the middle of the right side of the gel. I also don't know what those are.

Why are you running a polyacrylamide gel with TAE? Try TBE. It's possible at 200 V for so long your gel is getting too hot (the horizontal line might be a "melted" point - I know PA doesn't melt, but is it a weak point along the gel? Something weird happened there. Put your hand on the gel after a few hours - how hot is it? Depending upon your system, put a lot of buffer in the outer chamber to act as a heat sink, and/or run the gel in the cold room.

Another suggestion: run a MW marker. You can't really interpret a gel without markers, and how the marker runs can help you troubleshoot.

For a 300 bp piece you might try a higher percentage gel...or is this the point. I'm not sure what "heteroduplex" you're characterizing.

You can also place an ice pack in the buffer to keep the solution cold and place it on top of a stirrer with a magnet to disperse the temperature while it runs. Of course you'll have to change this out (I change them out every 45 minutes) just make sure to pause the voltage before you change it out.

I've never set polyacrylamide higher than 140v even with the ice pack. Typically only 120-130v once it's passed into the running gel. 

Also not sure if I am thinking of this right but ~300bp I would go 10%.

Also could the line be from the stacking gel? What were the reagents that you used to make your gel and how?

I've only done a few westerns but doing the above I've gotten good results. I usually am doing SDS-PAGE so I've never done TAE or TBE with polyacryl, that I usually do with agarose.

I think using 1X TBE buffer for preparing and running polyacrylamide gels is the best.

Also do not exceed 120 volts. You can try running a 10 % gel at 120 v for 12 hrs in the cold room.

Do you pre-run your gel for an hour or so to remove APS and TEMED? this also helps in proper running of polyacryl gels.

Lori,

I am loading samples twice since I’m trying to establish a protocol for a later experiment that will include running more than 2000 samples, so loading twice on the same gel will save time and gels. The line in the middle of the gel is my MAIN concern, why is this happening?

The shadows are really nothing; we by mistake dropped the ethidiumbromide on the gel prior to UV exposure.

The gel isn’t getting hot at all since we have a cooling system, so temperature is not an issue here.

Francesco,

We do not have any problem with the temperature since the system is connected to a cooling pump.

We have also ran the samples on a 8% gel and we get better separation for the heteroduplex.

We don’t stack the gels. We used 12 ml 40% Acrylamide (29:1), 0.8 ml 50X TAE buffer and added water up to 40 ml. Then we added 200 ul 10% APS and 25 ul TEMED.

Sumedha,

We have not used TBE so that might help. Also we have not pre-run our gels before, thanks for the tips.

Artur,

My main concern here is the continuous horizontal line that appears throughout the gel. The rest is ok and has worked fine. You see only a few bands since they are the only columns loaded with sample. We are setting up a protocol for a future run with more than 2000 samples, so we are trying to optimize the runs so we can load one gel at least 2 times.

We are going to run 8 gels at the time. We have enough equipment, but still a lot of samples, therefore we load twice on the same gel. I have even tried loading three times on the same gel and it looked ok for the second load but for the third load we got the same horizontal line, which seem to slow down the rest of the run.

I agree and disagree with different point raised so far:

TBE or TAE in my hands makes no difference.

I have never pre-run a gel but still I get good band resolution.

I don't use ethidium bromide any more - its toxic and a pain to dispose of. I find Gel Red is just as good.

I would be interested to know just how you decide on when to load the second set of samples: is the EBr in the gel and the buffer, so you remove the plate sandwich and view on a transilluminator, then load the next set if the first are out of the way? If so, why stop at three loads? I have done this with agarose gels, but the important thing seemed to be to rinse out the wells thoroughly before loading the next sample. The ionic conditions left in the wells might possible be generating your artificial "front".

But I agree that it is important that, for each set of samples, appropriate markers are loaded (which you may have to generate yourself by PCR - so that the "slow" bands in commercial markers don't interfere with subsequent loadings).

Would your bands resolve in 3 or 4% agarose? If so, there is a format of those gel tanks can allow 96 samples to be loaded at once. 

Geoff,

Thanks for your good comments.

After a few test runs we have now decide to load the second set of samples after 1,5 hrs run (with our new settings; 400V running during the day), and the third 1,5 hrs after the second load, and this works very well when we change the TAE buffer in between loads.

We do not have EBr in the gel (the 8% acrylamide gel consists now of 8 ml 40% acrylamide (29:1), 0,8 ml 50X TAE, 31,2 ml H2O, 300 ul 10% APS and 30 ul TEMED). Before taking a picture we incubate the gels in water containing EBr. We decided to load three times so we don't need to run so many gels (since we have almost 3000 samples to run) and with the new settings we see the bands of interest very clearly. We have also got rid of the horizontal line that we saw before (it looks like over using the buffer at high voltage caused the horizontal line).

Dear G,

Sounds very good. But I still encourage using "safe" Gel Red rather than "very hazardous" EBr!!

Good luck with your magnum opus!

G

Thanks a lot for your advice Geoff!

Ghazaleh