Why is my Agarose gel glowing? - (Sep/24/2004 )
I run my DNA samples in 1% Agarose Gel (UltraPure Agarose from Invitrogen) with 5microlitre of Ethidium Bromide (10mg/ml stock) added to it with TAE buffer. But while viewing the gel in UV transilluminator, I find the gel fluoresce gradually from bright to light. Though the bands are clearly visible, the photograph doesn't come well.
Can anyone suggest me why is this happening and how to get uniform stained gel?
Any help would be highly appreciated.
ethidiumbromide is a charged molecule and will move when in an electric field. so when doing you agarose gelelectrophoresis, your etbr moves also through the gel. so you get a higher concentration of etbr where is it moving and a lower concentration where it's moved away. that's quite normal when you put the etbr into the agarosegel. when staining the gel AFTER the run in an etbr solution will show - of course - no such gradients. staining is often recommended for the obeservation of small dna bands, as they will be much better to detect in contrast to etbr added into the gel directly.
in summary: you can stain your gel after the run, or you can lower the concentration of etbr in you gel and the effect will disappear or become less noticable, respectively.
it's probably not your gel that is not right. it's the camera. to get a uniform gel you have to have special functions on the camera like "zoom", "far", "darkness" ... But if you see nice DNA bands then don't worry about the uniformity of the gel on the photograph. your gel is ok...
some people suggest me to add EB directly to the TAE running buffer in the chamber instead of the one with gel. It proves great for me. And you don't need extra time to wait for staining. Just 3 times bigger amount than the one you put into the gel.
Which way the variation goes, from side to side or from end to end? You may also check your light box to make sure that the bulbs light evenly.
I think probably it's due to the amount of EtBr you loaded into the gelaas I have this problem before. The DNA is negative charged while EtBr is positive charged, that's why it seems moving towards the wells under the UV transilumination. I usually use the pipette tip's end to slightly touch the EtBr solution and that's enough to prepare 100ml gel slab.
I had that problem once. The solution to me was to shorten the apperture of the camera's diafragm. You must find the ideal apperture because the brightness of the bands change too.
Another problem that may be influencing the photograph is the fading of bands on the gel, this is due to the DNA being fragmented by the UV, and there is not a lot that can be done about it... my only suggestion is to keep the gel exposure time to a minimum, especially if you are trying to extract the bands for further analysis.
My solution is to cut down your EtBr use by 66%...i.e. instead of using 5 ul of 10mg/ml stock, just simply use 1.5-2 ul. I never really have a problem using very little amounts of EtBr.
Also, I notice that this gradient pattern usually becomes more frequent the longer you wait for the agarose to cool down before adding EtBr. I usually add my EtBr to my melted agarose when it is about 60 C...then I run away out of the lab before I breathe in any EtBr fumes. I don't know if there are such fumes, but I'm always suspicious of these things.
Hi , It may be because of high EtBR . BUT I had a similar problem once and finally realised that its the cellotape which is used to seal the gel casting tray, probably there are some chemicals that react with ETBR and migrate along with it on the gel... If you use a cellotape and the glowing is from fanit blue to flourescent blue then i am sure its because of the tape . What i do is aftre the gel dries i cut the surrounding and the glow vanishes. try this...