I am running agarose gel since very long and from very long time i face a problem with it. usually i made 20 ml for small and 40ml for large gel making. i add 1.5microlitre EtBr in 20 and 2 microlitre in 40 ml gel just before it solidifying temperature. then i mix it well with swirling for 1 minute. and then i pour it in a casting plate. load samples and run the gel.
when i terminate run and put the gel on transilluminator platform and switch on UV then gel showing two division on it. from upper adge to middle gel is shining with EtBr. means whole background and all looks like a fluorescence pink where the other half portion of gel is looking transparent colourless which is normal and advisable.
one more thing i encountered is that just in a half to one minute on transilluminator my bands start to disappear and within a minute they disappear. in starting these all problems were not encountered but since last 6 months same problems were encountered.
i have checked with new 1X TBE buffer, new EtBr and New agarose gel. but still facing the same problem.
is there any idea or solution for this problem then please let me know.
thank you very much.
Hello Jigar,
I am not sure I understood your problem fully, could you please attach a picture for reference? Also, do you know if your transilluminator is working properly, do other people in your group encounter similar difficulties?
you will often find this effect. ETBR runs up the gel ( opposite direction to the dna) so the bottom of the gel is dark and clear but the top is fluorescent. If you run the gel for a very long time all of the etbr will run out of the gel. If this is a problem just float the gel in a plate of 1xbuffer with etbr and the fluorescence will show in your bands again. The more serious problem is disappearing bands. If you put a gel on a transilluminator for too long photobleaching of the dna will occur. The dna will be nicked and broken and the etbr fluorescence will get weaker and vanish. This is paticularly effective when the filters which take out the short UV light on the transilluminator get old and start to let hard UV light through. The only thing that you can do is to take pictures very quickly and do not leave the gel on the filter with the uv lights on for any longer than absolutely needed. If you add some etbr to the tank buffer you minimise the half dark gel effect
flaw less answer given by Paul Rutland. yes I agree with him. we follow the floating of gel usually when we forget to add etbr to the gel before casting.
long term use of the uv transilluminator is not advisable. shift your daily practice to gel doc. instead of trans illuminator. while you are using a single instrument i.e. transilluminator, we can not say anything about the problem, use once a gel doc system after that if problem is persist than we can say about buffer, EtBr or agarose.
- Badges
- Science method