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southern blot problem - (Sep/29/2008 )

hi,

i hope someone can help me cause i am getting desperate. I have a serious problem with my southern blot. after hybridizing the probe i cant get ride of the radioactivity. instead of 200 cpm it is like 10000. i run a control probe on a different membrane and that one always works and i have no idea why my probe is not working anymore. once i got a really nice signal but i am just unable to repeat it. my probe is in a vector, it is cut out, gel purified and random primer labelled (katara ladderman kit) and hybridized. i know that my transfer, hybridization work but my probe gives me so much trouble. annoying.
someone has any idea? i would really appreciate it,
thanks

-paramo-

  • Did you remove the unincoporated radioactive dCTP after labeling the probe?
  • What is the formulation of wash buffer that you are using to remove the unhybridised radioactive probe?
  • What temperature are you washing? For how long?
  • Is the temperature in your hybridisation incubator okay? Can you check with a thermometer to confirm that the temperature is right.
  • Less relevant to this problem, make sure you clean the hybridisation cylinder. A clean cylinder is a good cylinder.
  • Are you using the right type of membrane? Not a western blot membrane but accident. Please check. Any undergrads in the lab? Or have you changed supplier or producer of southern blot nylon membrane.
  • Is the surface between the gel and membrane wiped dry? A wet, soaking gel, gives blurred bands... (Although this doesn't seem to be the cause here.)



Southern blot (like ligation) is a long winded process, so it is hard to sport the problem without being there watching it. Is there anyone in the lab who can help you do your experiment for you. And from there hopefully you can spot the problem. If not can you write, with every detail possible (temperature, time, what step) what you have done, and hopefully somebody here can help you

-perneseblue-


[*]Did you remove the unincoporated radioactive dCTP after labeling the probe?
[*]What is the formulation of wash buffer that you are using to remove the unhybridised radioactive probe?
[*]What temperature are you washing? For how long?
[*]Is the temperature in your hybridisation incubator okay? Can you check with a thermometer to confirm that the temperature is right.
[*]Less relevant to this problem, make sure you clean the hybridisation cylinder. A clean cylinder is a good cylinder.
[*]Are you using the right type of membrane? Not a western blot membrane but accident. Please check. Any undergrads in the lab? Or have you changed supplier or producer of southern blot nylon membrane.
[*]Is the surface between the gel and membrane wiped dry? A wet, soaking gel, gives blurred bands... (Although this doesn't seem to be the cause here.)
[/list]


Southern blot (like ligation) is a long winded process, so it is hard to sport the problem without being there watching it. Is there anyone in the lab who can help you do your experiment for you. And from there hopefully you can spot the problem. If not can you write, with every detail possible (temperature, time, what step) what you have done, and hopefully somebody here can help you
[/quote]



1. I ALWAYS USE A COLUMN TO REMOVE PRIMERS AND UNINCORPORATED P32. SHOULDNT BE THE PROBLEM.
2. I WASH WITH 2x SSC
3. IN GENERAL IT IS 2X5 MIN AT 65 BUT ITRIED 80 FOR 2 DAYS AND 0.1 SDS AND 0.5 SSC TOGETHER AND NOTHGING CAME OFF. AMAZING!
4. I CHECKED THE TEMPERATURE IT IS PERFECT.
5. THE ROLLING BOTTLES ARE ALWAYS CLEAN!
6. THE MEMBRANE IS CORRECT i KEEP IT LOCKED!;)

THE SOUTHERN BLOT IN GENERAL WORKS CAUSE I HAVE A POSTIVE CONTROL WHICH IS FOR THE ROSA 26 LOCUS AND ALWAYS GIVE ME A NICE BAND AND THE CONDITIONS ARE THE SAME, SAME MEBRANE AND SO ON. NEVER HAD THE PROBLEM THAT I COULDNT GET RID OF THE RADIOACTIVITY IN THE CASE OF THIS PROBE. JUST THESE 2 PROBES GIVE ME THIS PROBLEM.
BUT FINALLY I FOUND A PROBE WHICH SEEMS LIKE OK AT THE MOMENT AND I AM REPEATING IT I HOPE I GET A NICE BAND. AT LEAST I COULD WASH IT AND NOW I AM EXPOSING IT. CROSSING FINGERS.
THANKS A LOT!

-paramo-