Southern blot nightmare

Ed Stephenson estephen at biology.as.ua.edu
Thu Nov 10 18:25:42 EST 1994


In article <1994Nov7.101220.5277 at ucbeh> converrl at ucbeh.san.uc.edu writes:

>     I was writing regarding a problem I have with stripping a Southern Blot
>from a Magnacharge NT membrane:

>1. After probing this genomic Southern with a 2kb nick translated probe (100
>microliters of 3*10^5 cpm/microliter probe), my exposure shows that probe is
>hybidizing all over the blot in a random pattern (it looks like an inkblot
>test!). My boss told me that this type of background comes from not filtering
>the probe through a .45 micron Polysulfone acrodisc syringe filter prior to
>hybridization.

Check the effectiveness of DNase that you add to your nick-translation 
reaction. You can do a mock nick-translation (same amount of DNA, in NT 
buffer, but without label or polymerase), and run the product on a gel. The 
DNA should be extensively degraded, less than 1 kb (as detected on a 
natice gel, presumably the single-stranded MW will be less). In my 
experience you can get reasonably good incorporation in a nick translation 
without adding any DNase at all, but these large probes give a spotty or 
random smear background like you describe. Filtering the probe as your boss 
suggested may help, but I suspect you can improve the nick translation and 
get a good probe without having to filter.

Ed Stephenson



More information about the Methods mailing list