Pattern/cloud appearing on agarose gel
Posted 11 October 2012 - 12:39 AM
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.
Posted 11 October 2012 - 01:09 AM
Posted 11 October 2012 - 02:13 AM
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.
Posted 11 October 2012 - 03:47 AM
Posted 11 October 2012 - 03:47 AM
Posted 11 October 2012 - 03:53 AM
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.
Posted 11 October 2012 - 05:38 AM
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?
Posted 11 October 2012 - 07:45 AM
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?
Posted 11 October 2012 - 07:43 PM
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.
Posted 11 October 2012 - 08:02 PM
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.
Posted 11 October 2012 - 09:58 PM
Posted 12 October 2012 - 12:22 AM
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.
Posted 12 October 2012 - 04:36 AM
Posted 12 October 2012 - 05:30 PM