Very weird gel - I cannot explain these results (Oct/13/2010 )

The attached picture shows a gel that is supposed to be the result of a PCR reaction product @ 454 bp. This is a reaction that one of my undergraduate students set up. At first glance the smearing looks like the results of a typical DNA extraction with smearing at high molecular weights and even a band that might be mitochondrial DNA. Here's the weird part. Looking closely a the gel you can see the band wavering up and down in a distinct pattern across the gel. It's certainly not random. These are a dozen different human subjects using a typical PCR. If you notice, even in a single lane the band is often oriented diagonally within the lane. I have never seen anything like this. Can anyone help me explain this? I swear, I did not manipulate these results or picture (it's just a typical polaroid) in any way.

DrAnnie on Wed Oct 13 22:39:39 2010 said:

DrAnnie on Wed Oct 13 22:39:39 2010 said:

I believe you it isn't doctored (don't know why anyone wouldn't, why would anyone bother doctoring an image like that?)

I probably wouldn't worry about it and just repeat it. Too many weird things happen in the lab to follow each and every one up.

Chances are it is something to do with the gel or buffer (and the lanes look overloaded too). I was thinking maybe the gel was poured when it was too cool and had started to harden in some places already? But I don't know what that would look like? Or maybe the voltage was too high/ buffering capacity too poor and the gel heated up weirdly in places. I've seen diagonal bands like that before (particularly in large gels where I have run the product for a really long time), but the wavy thing is kind of cool!

Repeat the PCR with less template, run less product and make new gel carefully and run with new buffer.

Agree with leelee. I don't think this picture was doctored. It looks too real for me. I guess your student must be running a very dense and concentrated agarose, maybe up to 3%, and did not make sure the agarose fully melted and pour it only when the agarose almost solidify. Due to the EtBr was almost at half of the gel, I assume your student run it quite long, maybe with high voltage that constitute the heating on your gel as well.

But then, if you had figured it out how this happen, please let us know because I want to prank my supervisors with such a wavy gel... LOL

Well, I do know it was run on a 1.5% gel and at about 120V. Since the student had to leave, I took it out of the gel box. It was not melted. Without the waves, I would say that the student simply did not dilute the template and what you are looking at is essentially a DNA extraction with a mtDNA band. It's the waves that get me. They're so regular! That's why I suspect, as many of you do, that it has to do with the electricity in some way. I do not know anything about how the electrical component works in detail, so I was hoping someone with that kind of knowledge would say, "Oh, that's the so-and-so effect." I've been doing PCR's and agarose gels for 20 years and have NEVER seen anything like this. That's why I reached out to you guys. I did show an alumnus of mine who works with C. elegans and all he said was "I'd love to put this picture up in my lab! It's looks like a locomotion pattern!" Too bad I work with humans and beetles. Thanks for the input so far. I still await someone who has seen this before and knows exactly what's going on here!

Follow-up: Thanks to your input I did find out that our 10x TBE stock had a lot of precipitant which led to using higher than 1X concentration of TBE on our gels. However, that still doesn't explain the waviness to me. By changing the TBE we were able to get just smears with no wavy band (still not PCR product, but better than before). I officially give my permission for anyone to use my picture to freak out any supervisors or colleagues. Thanks for the help!

Thanks for the follow up and glad to hear that everything straightened out...literally. As for the no PCR product, I highly recommend you try doing this pcr in a series of optimization buffers that have various pH and salt concentrations. As long as the primers are good, I've always been able to get a pcr to work in at least one of the conditions. I'll attach a spreadsheet with my buffers which are all 10X by the way. Hopefully this will get your product. And thanks for permission to use the picture. A friend's lab always does a "scary data" competition for Halloween and this is a great entry.

it seems that your student used something circular to the gel to prevent it from floating ...this disturbes somehow the electric field and causes the weired pic in the end.

Regards,
p

may be the gel wasnt fixed into the gel bed while running...if the gel floats it might be moving up-down or sideways howmuch ever possible...so the dancing bands could be bcoz of the floated gel....

Gnana...