what is the maximum DNA I can load - Southern blot problem (Jun/20/2007 )
I am facing low sensitivity problem of southern blot. I have decreased the washing time and increased the hybridization time. But it did not change any. I used 10 ug per land. My question would it improve the signal if I know more DNA, and what is the maximum range?
A few questions:
Can you not detect your target sequence at all, or are the bands very faint? How long is your longest exposure and what does it look like? How much background is there?
This will help decide the best strategy to increase the signal.
For DNA concentration, you could increase to maybe 15 or 20 ug as long as you don't have to increase the size of the well to get it in.
You said you decreased the washing time, but the most important factors for stringency in washing are temperature and percentage of SSC. What is your washing protocol?
Hybridization with the probe should be increased to overnight if you're not already doing that.
You may have already checked these, but just in case: did you check that the specific activity of your probe is okay? Do you have a positive control and how much of the positive control can your probe detect?
You can debug your detection by spotting serial dilutions of your DNA sample on a membrane. If you can't detect the spot, then you won't be able to detect a southern band. This is much easier to debug and to quantify than going to the trouble of southern blots. Wait for those until your detection works. As others have said, SSC concentration and temperature are the primary determiners of washing efficiency.
i think washing time is also important like washing temperature. and if you use more than one membrane for washing together, be careful so that the membranes dont overlap each other