anyone got any idea how to make northern dot blots work? - I think it might be a probe problem (Jun/15/2006 )
I was wondering if you could help me at all. I have been given the job of setting up northern blotting as a new technique for our university to use to complement RT-PCR for quantifiying expression amounts, but I have having real problems. I have looked at all the booklets and protocols avalible but I can not get any response.
To start with I am looking at COX-2 expression by using my PCR probe (25bp) and PCR product (300bp) that shows expression for these RNA samples (cDNA). Because the RT-PCR works on these samples then I dont think the RNA has degraded. To test the probes and samples I am doing a dot blot first.
Ive attached the protocol that I have made up from various protocols but mainly the ones Amersham provide in their booklets (but cant help).
Can you have a look at them and see if there any abvious changes I can make, its driving me mad and as well as helping the science community it would save my sanity
Any help would be great
I'd start by making sure I can detect the probe on the membrane. Spot the *probe*, not the RNA sample on the membrane in dilutions, bind the probe to the membrane (UV) and go through the detection process. Don't bother with anything else until this works. Your problem is often that the probe is poorly labeled, and this will find that problem. Then, go on to the RNA. You might want to start with RNA which is not treated with glyoxal, to make sure that isn't the problem. Spot dilutions (along with a probe dilution control, if I were doing it) and detect with your probe + detection technique.
Cheers phage, I went through all that and realised the detection method was not specific enough, changed it to dig and did what you said and it has worked...............thanks loads.
I just need to try this with running a gel now!