southern blot hybridization - DIG - (Nov/19/2007 )
Hi,
I'm trying to do hybridization for a southern blot using DIG kit by roche and not radioactive.
with radioactive hybridization it's works perfect but I want to switch to DIG.
does anyone have any tips cause what I got from the company didn't help me at all.
I know that the probe is good and also the DNA but I just don't get any bands
please help !!!!! I don't want to use radioactive anymore
thank you !!!!!
How did you label the probe (PCR, random priming ...)? did you quantify it with a dig labeled standard? Can you see a background on the blots? If not, you can increase the concentration of the probe until you'll start to see a cloudy background after some hours (4-6, with CSPD as substrate) of exposition.
I have a little background, and I did increase the concentration of the probe. do you have any idea if the concentration of the target could matter (in the protocol they say small amount but i heard that high concentration of target could be better)
Probably, for big genomes, can be helpful to increase the DNA up to 20 micrograms. But I think the quality of the probe is more important. A PCR labeled probe giving a stong and distinct band, with a good shift in mobility due to the label respect a control PCR, is a good start. Then quantify it with a dig-labeled standard and use it at the concentration suggested by the kit.
There are many tips in the forum about this, and also check the dig manual and the FAQs in technical resources on the Roche web site.
[font="Arial"][/font][size="3"][/size]Hi, this might be a bit late but here is the protcol I used to use in my old lab. Works great!