15 replies to this topic

[Curtis]

It's about 3-4 months that we are having serious problem with our gels as a smear/pattern/cloud appears on top of the gel right below the wells. See the photo please. Does anybody know what this is? This time it looks like a painting!

It is like the pattern moves along all lanes and it is always at the top. The picture below is our PCR product. It doesn't matter if the TAE buffer is fresh or old, these patterns are always there.

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[bob1]

Is the pattern always the same or does it change with each gel?

[Curtis]

it changes, like clouds

[Astilius]

Now, I have never seen this before so I'm spitballing.
Is that ethidium bromide stained gels?   How are you making up the working solution of ethidium bromide?   Have you tried fresh stain?

Tell you what, it's very pretty.   Enter for the next Tate Prize.   I'd vote for it.

[phage434]

This looks as if it is an artifact of the gelling of the agarose.  I suspect that you have changed the way you are mixing or cooling the agarose before pouring the gel, and that some of it is setting more rapidly than other parts, leading to non-uniformity of the gel.  Make sure the agarose is well mixed and sufficiently warm before pouring the gel.  The solution should be crystal clear before pouring the gel.

[Curtis]

we tried fresh and old etbr, the cloud is still there. if you look at the photo the cloud is only over the PCR products. the ladder is clean.

[Curtis]

phage434, on 11 October 2012 - 03:47 AM, said:

This looks as if it is an artifact of the gelling of the agarose.  I suspect that you have changed the way you are mixing or cooling the agarose before pouring the gel, and that some of it is setting more rapidly than other parts, leading to non-uniformity of the gel.  Make sure the agarose is well mixed and sufficiently warm before pouring the gel.  The solution should be crystal clear before pouring the gel.

Thanks, I thought so too. we'll try. I never had this problem during my PhD. This problem started when I moved to a new lab.

[leelee]

I agree with phage434. This pattern you see is exactly what I've seen on the surface of gels that were poured too hot (and not well mixed) by a student in our lab recently.

Also, how much PCR product are you adding, and what is your template? Coz it looks overloaded to me.
Do you add the EtBr to the whole gel? Or to the samples themselves?

[Astilius]

Well, yes, it does look somewhat similar to the swirly patterns you get in a gel that's been poured too hot but I'd be concerned about the fact that the sample lanes are lighting up in their entirety.   I'm not sure that pouring a gel too hot would cause all that's being seen, You more often get deformations in bands and that's not happening.

Have you tried running PCR product that you know is very good (maybe from a sister lab) and comparing how your gels look?
Have you had someone else pour a gel?

[Curtis]

I attached two more gel photos below. The first gel is digested plasmids. We already know if the DNA concentration is too high the bands won't look good, but in here we loaded equal amount of plasmid in each well. The gel looks like a painting of a night with clouds and stars.

The second gel is for different samples: digested pCIneo which is cut from the gel, undigested plasmid, and some other purified PCR products. You can't see the cloud in gel purified PCR samples. But clearly there is something stock in the wells of digested pCIneo.

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[leelee]

The other time I've seen something like this happen is when photographs are taken of the gels when they haven't been drained very well, or the transilluminator not wiped down after the last gel was on it. Although it does't really explain why in most cases you only see it in your loaded lanes.

Just a thought anyway.

When you say you loaded equal amounts of plasmid to the first gel- do you mean volume? Or amount of DNA?

Edited by leelee, 11 October 2012 - 08:02 PM.

[Curtis]

I mean ug. We normally rinse briefly with water right before putting it on transilluminator, but I agree that the second gel photo I sent is not rinsed well. The cloud is inside the gel. I doesn't go away no matter how much I wash it with water.

[Astilius]

Those "stars" in the second gel - they look like crud.   Maybe dust and bits of old tissue.
What sort of water are you using to make gels up in?   Have you tired making gels up in something like milli-q water just to see?  
You are cleaning out the flask to make gels beforehand, right?   Right?

Try this:  make a gel up with a new flask, using new bottled water and fresh agarose.   Hell, just new everything but make sure you have new flasks and water.
Vessels and water seem to be something people overlook as a source of contamination.

I'll say this next bit, though from most of the bands I don't think this is an issue, so please don't take it as an insult:  when boiling the agarose, make sure that it does boil a little and then swirl to ensure homogeneity of the melt.   Then cool so that you can hold it comfortably in your hand (not too hot, not too cool), add your ethidium bromide (unless you stain the whole gel afterwards in a tank of stain) and swirl until it's homogenous.  


Pour the gel in a super-clean tank (clean it, then clean it again and then rinse it in the purest water that you have - a new box/bottle if possible).
My hypothesis is that your lab needs a clean and/or the cleaning regime for at least one of your components, be it glassware or tank apperatus has some sort of carry-over on it.   You could just have a mucky-pup of a tech in there.

If possible, make a gel in a sister-lab and compare results.

[phage434]

Would you please take a picture with no gel in your gel imager?  I think there is fluorescent material on your imager plate.  No fair cleaning it first.

[uday]

Since your ladder is pretty clear, there should be something wrong in your PCR master mix or sample. Try changing the master mix !