S.Blotting - (May/29/2008 )
Hello all I am new to Southern blotting. My experiment involves digesting genomic DNA - human with various enzymes - NEB. My first problem is I digest the same amount of DNA with same amount of enzyme separately. But after digestion, I speck the DNA using nanodrop, I don't get all in the similar ranges. Why is this so? for ex: I digest 12ug of DNA with EcoR1. Xba1, Hind III etc seperately - the total reaction volume is 50ul. So I would expect around~ 240ng/ul. But what I get is some would be high and some others as low as 80ng/ul. What could be the problem? Thanks for your replies.
I hope I might be able to provide some help....but I rarely spec the DNA after the enzyme digestion. Keep in mind that you are allowing these digestions to occur in a solution containing protein (your enzyme) at a decent concentration and potentially BSA. All these things could throw your spec readings. As such, I always record my INITIAL concentration of DNA. You can precipitate your DNA if you need to prior to loading onto a gel (only if you need to concentrate the volume amount) and test a small aliquot to ensure that you see good digestion of your starting material (I highly recommend this as it is important to ensure your enzymes have cut thoroughly).
I've done a great deal of southern blots and have never had a problem with big loses in DNA...but the enzymes failing to cut well, that's another story!
Hope this helps.
Hallo, just day ago, there was a girl from next door lab. Nanodrop measure starange quantities of the DNA to her. I had the same experiences with this instrument and several frends as well. You can try to run 1ul from all samples on ELFO to see the concentration is diferent(best way is to do 2 fold dilution fore time)