southern blotting problem with DIG - southern blotting problem with DIG (Nov/23/2008 )

hI there
im new to bioforum...but really need some help with southern blotting as im new to it as well.....i am not getting any signal with my southerns....not even my positive control or markers show any bands in the blot..i just get a blank blot.
my probe is a pcr product (700bp)..i have labelled with dig...checked the labelling efficiency. I did dotblotting ...where i added about 4 ul of dna directly into the membrane and fixed it and hybradized it with the probe at 37 degrees, and stringent washes at 65 degrees. I got results from dot blotting so i tried the same thing with southern blotting as i need to know the copy number of my gene of interst. But when even i do it i just do not get any result at all...no bands....just a blank blot.....i am so frustrated. i am giving the conditions i followwed
i used 10ug g.dna , digested with res enzymes....n ran it on gel 5-6 hrs...wash it in depurination buffer for 10 min....wash in denaturation buffer 30min.....n in neutralisation buffer for 2 x 15 min. set up a southern blot o/n.
next day i take the blot...take the side which is facing the gel n place it on blotting paper ....fix it in oven at 80 degress for 10 min and then uv fix it 2-5 min.
prehybradize the probe with dig easy buffer (prewarmed at 37 degrees) for 30 min ....hybridize the blot overnight at 37 degrees with the hyb buffer (40ul probe was taken , denatured at 95 degree for 5 min and rapidly cool in ice, and added to 40 ml dig easy buffer).
next day i perform the stringency washes at 65 degrees....and rest ofthe washes at 40 degrees and expose in the imager for 30min .
i get no bands at all.
PLEASE HELP ME. wHERE CAN I GO WRONG????
IS it during the transfer ......ie after dismantling the blot i keep the blot in the blotting paper to dry ....does the dna stick to the blotting paper as it is not yet fixed?
am i not using right hybradisation temp.....37degree>???? am i using too much of probe....but when i do the dotblot i use 2ulprobe in 2 ml digeasy buffer and it works fine....but now its doesnt.................
PLEASE GIVE ME SOME TIPS....if my poritive control doesnt work theres no way my samples will work .....ideas please!!!!!!!!!!

You need to track down where the DNA is being lost or not detected. Start with a serial dilution of your positive (uncut) DNA sample, spotted on a membrane, and follow you existing protocols of binding, UV, hybridization, and detection. Figure out the minimum detectable amount of your positive sample that works. Try reducing the wash stringency (higher salt, lower temp) until you get good (or any) detection.

Then move on to your gel, if you have not localized the problem. Make sure you are using membrane (and not the paper between membranes! -- sorry, just checking). You could spot serial dilutions onto a blank gel and blot the fake gel onto membranes. Overnight may be too long to blot -- a few hours probably is enough.

I take it you are using a DIG labeled marker/ladder? If this isn't showing up them there is something wrong with either the transfer or the detection processes.

Transfer: Make sure that your stack isn't "short circuiting" i.e. touching the bridges so that the buffer doesn't have to go through the gel to wet the paper. Do you have enough weight on the top? - 200-400 g should be enough. Are you using the right buffers? What type of membrane are you using - nylon or nitrocellulose? If nylon, UV is enough to crosslink, no need to bake. 2-5 min seems a bit long to me, I do 1 min in a standard gel imaging cabinet. You could be degrading your DNA by causing breaks in the strands.

Detection: Make sure your antibody is stored properly - aliquots at -20 is fine. Are your luminescence reagents freshly prepared? Try doing a longer exposure: my single copy detections often took 2-5 hours exposure with film, which is more sensitive than electronic devices in my experience.

Have you calculated your hyb temp using the formula given in the manual? If so it should be OK.

I've been experiencing the exact same problems with the DIG system as you have been experiencing; and I have been struggling with this system for the past 6 months with little to no success with genomic DNA; it works wonderfully for plasmid DNA.

Here are a few things I've tried:
- check if DNA has transferred: after transfer, I place both the gel and the membrane on a UV transilluminator. If transfer was complete, the gel should not fluoresce and the membrane should faintly fluoresce.

- make sure everything is ROCHE: I know this seems a little obvious, but apparently ONLY Roche positively charged membrane works well with the Roche anti-DIG system; all other brands give high background

- lower the stringency of your washes: start with 2 low stringency washes (2X SSC 0.1% SDS @ 42oC) then follow with 2 med/high stringency washes (0.1X SSC 01% SDS @ 60oC)...starting with the 65oC wash may strip off all hybridized probe and thus strip off any chance of obtaining signal with the anti-DIG antibody

- increase the concentration of the anti-DIG antibody to 1:10000 or perhaps even 1:5000 in no more than 20ml/100cm2 of membrane

- make sure your anti-DIG antibody has not expired; if so, order some new antibody

- use the CDP-Star ready-to-use substrate for your ECL detection step: this substrate should yield signal within 1-3min for genomic DNA; CSPD takes much longer to develop a signal; for plasmid DNA, you should get signal within 10-30sec

I'm starting to think it might be a copy number problem; but this system purportedly can detect single copy genes. I've checked my genomic DNA with PCR using the same primers I used for my probe, and the sequence is definitely present, albeit in low quantities. So the southern HAS to work, but, alas, it still isn't.

Well good luck to you with your Southern/anti-DIG saga...if it still doesn't work let's go grab a beer and get depressed together. LOL!

cheers!

- You could also try increasing the amount of digested genomic DNA you start with to 15ug.

- Hybridization is also optimal at 42oC overnight.