Northern - really stuck here! - (Jul/03/2008 )
Ok so I have been doing Northerns for a while but have failed to get the results that I am after.
The problems appear to be with my probes because the control actin probe which comes with the Strip-EZ DNA kit works perfectly and I get a beautiful signal within 30 minutes!! To get my own probes, I make cDNA from mouse RNA and then PCR up my region of interest using the cDNA as a template. I get great PCR bands. I then phenol:chlroform clean my probes and label with 32-P using Ambion Strip-EZ DNA kit. When I check the incorporation, it is not great but is workable (around 50-60%). I run a blot of mouse RNA usually not going above 10ug. I use standard wash conditions and the same ones that I used for the control.....but NOTHING!!!
I have tried probes of all length ranging from 200-1500 bp. I have no idea what is going on. I am usually very impatient and write the blot off as a failure if I do not get a signal after 1 day. Should I just leave the film down in the freezer for longer?
Do your probes have the same annealing temperature as the control probes? I am guessing that is the major issue, try dropping the annealing temp down a few degrees.
I suggest switching away from radiolabelled probes and go for DIG labelled, can PCR incorporate into the probe and will give great signal in a couple of hour exposure, no more shoving cassettes in the freezer and playing with Geiger counters. Also the DIG manual is really good.
The problems appear to be with my probes because the control actin probe which comes with the Strip-EZ DNA kit works perfectly and I get a beautiful signal within 30 minutes!! To get my own probes, I make cDNA from mouse RNA and then PCR up my region of interest using the cDNA as a template. I get great PCR bands. I then phenol:chlroform clean my probes and label with 32-P using Ambion Strip-EZ DNA kit. When I check the incorporation, it is not great but is workable (around 50-60%). I run a blot of mouse RNA usually not going above 10ug. I use standard wash conditions and the same ones that I used for the control.....but NOTHING!!!
I have tried probes of all length ranging from 200-1500 bp. I have no idea what is going on. I am usually very impatient and write the blot off as a failure if I do not get a signal after 1 day. Should I just leave the film down in the freezer for longer?
I would definitely try leaving the film down longer. It's been a long time since I've done northern blots, but I recall at times leaving the film down for days if not weeks. If your transcripts are not very abundant, then you won't see as good of a signal as actin (remember actin is very abundant). Perhaps if you have available, you could also try running a control such as a construct where your gene of interest is overexpressed. That would help you to determine if your probes are working.
The problems appear to be with my probes because the control actin probe which comes with the Strip-EZ DNA kit works perfectly and I get a beautiful signal within 30 minutes!! To get my own probes, I make cDNA from mouse RNA and then PCR up my region of interest using the cDNA as a template. I get great PCR bands. I then phenol:chlroform clean my probes and label with 32-P using Ambion Strip-EZ DNA kit. When I check the incorporation, it is not great but is workable (around 50-60%). I run a blot of mouse RNA usually not going above 10ug. I use standard wash conditions and the same ones that I used for the control.....but NOTHING!!!
I have tried probes of all length ranging from 200-1500 bp. I have no idea what is going on. I am usually very impatient and write the blot off as a failure if I do not get a signal after 1 day. Should I just leave the film down in the freezer for longer?
I would definitely try leaving the film down longer. It's been a long time since I've done northern blots, but I recall at times leaving the film down for days if not weeks. If your transcripts are not very abundant, then you won't see as good of a signal as actin (remember actin is very abundant). Perhaps if you have available, you could also try running a control such as a construct where your gene of interest is overexpressed. That would help you to determine if your probes are working.
OK so changed the annealing temperature and Im not getting anything specific here (as expected really)!!! This is really annoying. I know the gene is expressed to pretty decent levels in this tissue so it should be coming up fairly easily with an intensifying screen. Seems the specific activity of the probe is just not good enough. Any ideas on how to beef this up a bit?