Hi Stephanie,

we never got the DIG system to work with Northern blots. The only option was to use radioactively labeled probes (alpha-32-P). Maybe other members have better experiences?

Good luck!

Christian

Hi Stephanie,

radioactive-labeled oligos are way more sensitive than digoxigenin-labeled probes, but DIG should work for sRNAs that accumulate to high levels. Also, maybe an stupid question, do you transcribe your sRNA probe or do you use a T4 ligation method? If you transcribe your miRNA complement you should check how many "A's" does it have.

I think that two of the main things that you could improve is using EDC crosslinking and regulate your hybridization temperature. You can potentially re-use your membrane (previously stripped) and Dig-labeled probe. That can save your lab some money while you optimize the protocol. Of course, to a certain extend. I have re-used probes and membranes up to 4 times.

Best,

German.

Hi German,

I am not sure I understand the question. (2nd year PhD student here- still trying to get a grasp on everything!) My lab's former student (long gone) used primers for his Northern blots that he developed from Biology Workbench and built/purchased through IDT. But he used the radioactive method.

I'm already using EDC crosslinking and have regulated the hybridization temperature per the original paper's specifications.

The only difference has been the probe concentration. Without LNA probes, I didn't use the same concentration of probe and used the amount normally used in the radioactive protocol (specifically, using 0.5ul primer stock at 40mM vs 2 ul of 50pmol LNA probe).

Hi Stephanie, I am just wondering if you have solved the problem with DIG labelled probes. Have used LNA probes? I am also going to try the DIG labelled probe protocol for the northern blotting, so I am just curious if you have finally managed to perform northern blotting with DIG. Thanks in advance.

I have been using LNA probes. We have had a lot of trouble with several of the steps. But we think that one of the issues was that Li-Cor equipment doesn't like to work without Li-Cor specific probes (per another researcher) so now that we are using X-ray film, I should be able to let you know this week if it works or not!

hey Stephanie and Steffen! I am also stuck with DIG based northern blot. I can get the DIG-labelled molecular weight marker on the blot but not any other specific band. when I perform dot-blot hybridization, that gives me positive results suggesting labeling reaction is a success. any help in this regard would be appreciated.