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troubleshooting northern blot - (Apr/24/2007 )

hi

i have done a northern blot and in the last step , there was no result on the film (thr was no background as well).

Experiment was repeated and the same prob occurs.

Have checked the membrane, probe, dig and chemiluminescent and its working fine.

even view the membrane transfered w RNA under UV and there are bands.

tot that something must have gone wrong during hybridization. can anyone advise?


thanks

-heidi-

Spot serial dilutions of your probe onto a membrane, and detect those. Only when that works, go forward.

Then, spot serial dilutions of your DNA sample onto a membrane, along with the serial dilutions of the probe. Hybridize with the probe, and detect. Only when that works, go forward.

Finally, run an RNA gel, blot, spot serial dilutions of both your RNA sample and probe, detect, and enjoy your success (along with your detection sensitivity controls).

-phage434-

QUOTE (phage434 @ Apr 24 2007, 09:01 PM)
Spot serial dilutions of your probe onto a membrane, and detect those. Only when that works, go forward.

Then, spot serial dilutions of your DNA sample onto a membrane, along with the serial dilutions of the probe. Hybridize with the probe, and detect. Only when that works, go forward.

Finally, run an RNA gel, blot, spot serial dilutions of both your RNA sample and probe, detect, and enjoy your success (along with your detection sensitivity controls).



i did the spot serial dilution (as attached) but it still din work out. there was no background on the membrane as well.
any other possibilities?

-heidi-

QUOTE (heidi @ May 2 2007, 01:56 PM)
QUOTE (phage434 @ Apr 24 2007, 09:01 PM)
Spot serial dilutions of your probe onto a membrane, and detect those. Only when that works, go forward.

Then, spot serial dilutions of your DNA sample onto a membrane, along with the serial dilutions of the probe. Hybridize with the probe, and detect. Only when that works, go forward.

Finally, run an RNA gel, blot, spot serial dilutions of both your RNA sample and probe, detect, and enjoy your success (along with your detection sensitivity controls).



i did the spot serial dilution (as attached) but it still din work out. there was no background on the membrane as well.
any other possibilities?



As a positive control you can run the gene for which you are making the probe, and then procede usually as you do, and if you get signals in this was then you can be sure that the method is working. also u can use marker in the and make probe for marker also.

hope it helps

-coffee-

In what sense did the dot blot not work out? I see spots on both your probe dilution and on your DNA dilution. You might ask for higher sensitivity on the RNA dilution series, but there is certainly some detection. You can now work on optimizing the sensitivity by controlling blocking, hybridization conditions, washing conditions etc. You can estimate the sensitivity you require to detect the RNA you must detect. When you have reached that level, you can try a blot.

-phage434-

QUOTE (phage434 @ May 3 2007, 11:25 AM)
In what sense did the dot blot not work out? I see spots on both your probe dilution and on your DNA dilution. You might ask for higher sensitivity on the RNA dilution series, but there is certainly some detection. You can now work on optimizing the sensitivity by controlling blocking, hybridization conditions, washing conditions etc. You can estimate the sensitivity you require to detect the RNA you must detect. When you have reached that level, you can try a blot.



sorry what i meant was that the hybridization stil din work out (after making sure that the probe is fine using dot blot serial dilution). The x-ray film does not show any bands and there was no background noise (compared with film that works, usually can see edges of the membrane in the film). The film is fine cos can see marks from light exposure at the edges.

checked membrane under UV, the blotting was successful, RNA is on menbrane. so kinda puzzled what might have went wrong.

thanks in advance

-heidi-

Well, maybe I'm not interpreting your dot blot properly. Is the right hand set of dots serial dilutions of your RNA sample (the same RNA that you would run on a gel)? Are those spots a result of hybridization with the probe?

-phage434-

QUOTE (phage434 @ May 4 2007, 10:18 AM)
Well, maybe I'm not interpreting your dot blot properly. Is the right hand set of dots serial dilutions of your RNA sample (the same RNA that you would run on a gel)? Are those spots a result of hybridization with the probe?



hi
u were right initially. the right side is my dilution of DNA controls (not the result of hybridization). Comparison with probe was done using the DIG
labelling kit.

DNA fixed to membrane was incubated, added with antibody, CSPD and finally captured.

so as far as i think ,probe should not b the cause to the failure of hybridization rite?
what could i alter/monitor in controlling blocking, hybridization conditions, washing conditions to troubleshoot which is the culprit?
could temp, time or buffer be the cause of this failure?

-heidi-