Why is my Agarose gel glowing? - (Sep/24/2004 )

HI
the best method to stain ur gel with et br willbe to strain it after the gel run is over. also u can try using lesser concentrations of etbr and stan them for a small duration to get sharp view of the bands. erbr will move through the gel if u are loading it along with the along with the buffer.
regards
madhan shankar
HOD biotech
COimbatore
india

First thing first..... The gradation which you see is due to the movement of EtBr in the gel...EtBr is +vely charged so when you run the gel, as DNA moves from the -ve to the +ve, EtBr moves from +ve to -ve electrode....thus you see a gradation being formed after you run your gel from 40 minutes to 1 hour......

EtBr contains a planar tricyclic phenanthridine ring system due to which it is able to intercalate between the staked base pairs of double-stranded DNA..... It doesn’t matter if you put EtBr in the gel itself or you stain it afterwards or in the running buffer.... It will intercalate anyhow.....

But the only way you can remove the gradation is by staining the Gel after you run it, because that will be even staining ....

One disadvantage of putting EtBr straight in the gel is that the mobility of dsDNA is also reduced by about 15% in the presence of EtBr.....

You can easily stain it after running by putting your gel in a buffer solution which contains EtBr at a concentration of .5 microgram/ml....You can even reuse this stained buffer for quite some time...But be sure to maintain this concentration of EtBr.....

You can also enhance fluorescence by destaining the gel in a solution containg 10mM Mg++ before examining it in UV......

Hope this solves your problem.....
Regards,
Veenu
B.Tech,
Biotechnology.

Hi There! Just run the gel for a longer time (if u'r product is big enough) and that will take care of the problem!!