Sothern Probe Design principles - (Jul/12/2007 )
Hi there, I want to desing a Southern probe. I know the longer is the better principle, but what else should I keep in mind when designing a probe? (GC content, starting/ending bases, etc)
it depends what you want to hybridize it with. If your DNA on the blot is genomic DNA, make sure your probe does not contain any repetitive elemens (like LINEs or Alus) otherwise the probe will hybridize everywhere. For the rest, I usually take a probe of app 700 bp, and I don't really look at GC content and such.
Thank you. I started the Southern, and though from the positive controls (plasmids from which the probe was amplified) I got the appropriate bands labelled, I got only a smear from genomic DNA. After a long exposition time (with Roche DIG detection kit) also appeared the non-labelled bands in the plasmids. In the genomic, I have only one sample which shows aome really fade bands, but not only the specific ones. Do you have any idea what can be the problem? Probve specificity, or digestion...? I digested my samples (both genomisc a plasmid) overnight, and before loading it to my Southern-gel, I ran them on a minigel with EtBr. I saw the expeceted bands in the plasmids and I saw a nice uniform smear for each lanes of genomic DNAs so I thought the digestion was ok. The transfer went on ON, it seemed to be ok, too (thogh of course I didn't put EtBr into it, but the gel was really thin by next morning). In some gDNA lane I can observe a strong color on the side but it is not uniform to the gDNA samples. Any ideas are appreciated.
i think DNA quantity in you sample is lower so you get a fade band as a signal. increase your starting DNA amount and try again.
and also for southern your gDNA must be very clean. after transfer DNA to the membrane check the gel under UV ray is the DNA remain in the gel or properly transfered.
Thanks. I think my gDNA is quite clear, we clean it on a Qiagen column and measuring with Nanodrop shows a high purity of DNA (OD260/280>1,95).
What I'm much more worried about is that I got some unspecific fade bands while in BLAST I din't find homology of my probes with other regions of the gDNA... however, Il check it again right now.
i generelly try to use probes for southern that span inside genes (where is that possible), because they work with great spesificity, and are about 300-500bp.
however, i wanted just to add that i had trouble trying to work with the DIG of Roche....maybe its just me....but just have it in mind.
I find better the Amersham ECL system for southern.