no bands, no ladders, nothing at all in my gels - (Mar/24/2008 )

okay, let's see if anyone can solve this big 'mystery'! this is a very long message. apologies.

i ran a gel with three rows of wells that were all loaded with PCR product. when I looked at the gel under the UV, I noticed a black cloud running through the gel. it wasn't exactly a cloud. it was actually shaped like a hand with the middle of the palm at the top edge of the gel and the fingers pointing towards the bottom of the gel. it was like a shadow of someone's hand on my gel. but the strange thing is that where the shadow lay, the gel was completely black with no sign of any flourescence.

for example, the first and second rows of wells which would be covered by the palm of the hand show no bands whatsoever. that part of the gel was completley black. not even the KB ladders ( i loaded three of them in each row of wells) showed up. in the last row of wells, where the fingers were, bands only showed up between the fingers but the gel was black where the fingers would be. some bands were half under the fingers and half outside of the fingers. these bands were black under the finger part but were bright where they weren't. it was like they were cut in half.

so I restained it in EtBR for fifteen minutes but that did not help.

I repeated the gel electrophoresis and this time the entire gel was black.

Then I ran two more gels using completely different PCR products and they worked fine.

I then ran yet another gel, again with completely different PCR products, and this gel also came out completely black.

Today I repeated the electrophoresis using the products that came out black and it worked this time!!

So, can anyone suggest what may have happened?

Only two people have been able to offer a suggestion and they both say it must have been an air bubble underneath the gel that did not allow the products to migrate in the gel or that the surface of the gel was exposed to air because the buffer in the gel electrophoresis chamber probably evaporated. But I disagree, because I don't think air would be in the shape of a hand with fingers. Also, I think an air bubble would be very visible and I would have seen it. And, if that were true, wouldn't the ladders and the PCR products stay in the wells and show up under UV? All the wells were black.

Maybe the gel floated while I did the electrophoresis? Would this affect the gel in such a way that it would appear black under UV?? By the way, I changed the buffer and increased the EtBR concentration and this did not help. The buffer is 1x sodium borate.

I apologize for the very long message, but this has me scratching my head so much that I have a bald spot!

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Sounds strange! But, isn't science really strange (atleast in our hands!)...

1. Do you have pictures of your gels that you can upload here? It probably will be good to see actually what you have.
2. Have you checked that you have the right setting in your camera i.e., EtBr detection as opposed to SYBR green detection or something else. I once had a gel that was completely void of any bands because of this.
3. Have you tried SYBR green staining of your gel as opposed to EtBr?
4. Are you sure that your 'hand' is not really the gel loading buffer?

Good luck...

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I think this can be classified in the X-files of molecular biology.
If I were you, I would change whatever I'm using, e.g. TAE, loading dye etc, and start over again. Just have to make sure that you trust all the stuff you're using. Also, change the buffer that's still in the electrophoresis set. Make sure EtBr is not contaminated with something.

Anyway, hope that it's not the UV bulb that's burnt. There're normally two to three bulbs and if one is burnt, you'll see a darker region on your gel. Also make sure that the camera lens are clean etc.

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here is a picture of the gel. my memory isn't all that good. now that i look at it i see four rows of wells and the black region does not really look like a hand.

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this is a picture of the same gel after i placed it back into the EtBr for further staining.

after this I changed the EtBr solution and the buffer in the electrophoresis chamber. i ran two other gels and they came out fine but then a third one came out all black with no bands or ladders.

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1. i loaded a couple of pics

2. i think the camera was in the right setting. i can't say for sure. but the first pic, i don't think had anything to do with the setting.

3. we only use EtBr. again, if you look at the pics you'll see that this is not the problem. as for the third gel that came out completely black (no pic, sorry), it came out okay after i reloaded yet another gel with the same pcr products. why the one came out black and the other okay is a mystery because i used the same EtBr solution but changed the electrophoresis buffer. however, previously i used the same buffer and one gel came out black and the other was fine.

4. see number three.

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did you stain the third gel? did the bands show up?

it sounds like the etbr in your buffer is getting depleted during the runs. since etbr migrates (in the opposite direction of the sample) this can be expected (unless you remix your buffer after or during each run).

you can either increase the amount of etbr, remix or replace (more frequently) the buffer.

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actually, the staining and gel run are done separately. there is no EtBr in the electrophoresis buffer, just sodium borate. i threw out the third gel and reran the same pcr products on a different day in a different gel and the bands came out nicely.

i don't know, this is just one big mystery that i don't think will ever be solved. it's really the first picture of the gel with the black "fingers" that i find most mysterious. the other gels came out completely black so it's easy to say it was the buffer or etbr solution or UV light problems, etc. for those gels. but for the gel in the first picture, i think the mystery will follow me until my last breath...............

unless, of course, i experiment and try to replicate the mystery. i might do that one day.

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