southern blot expert needed - How do you improve your southern? (Apr/22/2005 )
I've been struggling with genomic southern blots for a while now and I'm still getting very weak signal (I use 32P labeled probes).
I've read many southern protocols and my electrophoresis and blot seem to be done according to the standard technique.
I synthesize the probes myself (regular PCR), and I randomly labeled them with 32P using a kit from Stratagene. I tried a couple of other kits but this one showed to have the best efficiency.
I've tried hybridizations at different temperatures (from 42 to 68 oC), always using the hybridization solution from BD (I never tried the "homemade" one for these genomic southerns).
So, I have DNA in my membrane, which I crosslink, the probes (sizes vary, from 500 bp to 1.2kb) appear to be quite hot, but still my signal (when get one) is extremely weak.
Does anyone have some ideas on how to improve my southerns?
Thanks in advance!!
some questions for more information:
are your probes to single copy regions?
How stringent are your washes?
Have you tried spotting some unlabelled probe on the membrane to test if the labelled probe hybridises to this?
Do you check the radiation signal after each wash?
recently i've done two improvment for my blottings
i purified the labelled probe
i change the ratio of radioactive nucleotide and probe to label (nucleotide moles need to be at least equal of amount of probe to label for a 5' terminus labelling). Maybe this ratio need to be improved in your experiences?
I do purify my probes... As to the ratio radioisotope/amount of probe to label, using the kit should make the reaction less sensitive to those variations, I think.
Q1: If I understood your question properly, the answer is no. My probes are suppose to hybridize to both copies, ie both alleles of the given locus.
Q2: Hum... that's a good point. I do 6 washes of 10-15min each, with: 2X SSC (at RmT), followed by 1X SSC and 0.1X SSC at 60 oC. So, it's 2 washes with each one of these solutions. Do you think the last two washes, which are high-stringent, could be removing too much probe?
Q3: I haven't tried that, but one time I did run 1uL of my unlabeled probe along with my DNA samples of interest and the signal was very strong in that lane. That was also a good control for me to confirm that all the other steps (electrophoresis, bloting, etc) seem to be fine.
Q4: no, I usually don't do that...
i assume if the targeted region is alleles, it's a double copy region. That means signal will not be diluted.
i did that too with my first protocoles and i still get signal. But nothing except non specific. So i would not strongly consider a positive measurment.