Hi Luca

Regarding your question about electroblotting, I had experience in northern blots for miRNA and small non-coding RNA analysis. In this case I used denaturing acrylamide gels for the RNA separation and a common semi-dry device from Pharmacia (or Amhershan, I cannot remember) for electro-blotting. The membranes were, as Thomas already pointed out, Hybond-N+. This will help you because of the positive charge of the nylon membrane. Transfers were made in TBE 0.5X buffer at a constant current of 1 mA per square centimeter of nylon membrane during 1 hour, with excelent results.

I am adding an attached document with a protocol from the UTexas.

I have no idea if it is possible to transfer agarose gels by electro-blot.... :(

Good luck. Best

Paco

http://www.utexas.edu/research/papoulaslab/protocols/molecularbiology/miRNA_Northern_Blot.pdf

I used to run the procedure similar to Thomas Schwarz above using the Turbo Blotter. This system works well, just takes overnight and like all transfers, you need to make sure that there are no air bubbles and that you have enough buffer. I am pretty sure we used nylon membranes, but it has been over 10 years since I ran a Northern blot. Make sure your samples are well denatured and that you cross-link the RNAs to the membrane and use a good hybridization buffer. I don't think I ever came across anyone electrotransferring samples out of agarose. I think this may be one of those times that old-fashioned ways win out and you just need to wait on the transfer.

Good luck.

Jason

My experience with Northern blotting using the capillary method is quite positive. You should use positively charged nylon membranes.

This depends on your gel. Have you tried running your RNA

In user-friendly glyoxal agarose gel?

Regards,

Aziz

In my experience the capillary method works best and I always got more than 90% transfer for even longer transcripts of about 10 kb or more. I generally denature the RNAs, quick chill and add little bit of EtBr (about 0.2 ul of 10 mg/ml stock per 25 ul RNA solution) and resolve on the gel. I always used Ambion® BrightStar®-Plus Positively Charged Nylon Membrane and this works best for me. By adding EtBr you can literally check the RNA on the blot under UV. Dont not add EtBr before denaturation.

good luck!!

I do Northerns out of agarose gels pretty much every day. I used to do capillary transfer, but never again, it takes forever. For my electroblotting, I use a semi-dry trasfer cell from Bio-rad (Trans-Blot SD). Never had a problem or melting gels. As far as a protocol what I do is pretty simple. First you should soak filter paper in running buffer, for this I use running buffer without formaldehyde so I don't contaminate everything. Once I do that, I put several layers of the filter paper on the bottom plate of the transfer cell, this would be the positive end. after that I put the membrane (previously soaked in buffer too). Using a pipette I get rid of the bubbles. The best way is to use a rolling pin motion. Then I place the gel over the membrane and again I get rid of the bubbles. Finally, on top of that I add a couple more layers of buffer soaked filter paper and again use the pipette to eliminate air pockets. I run that for 30 to 45 minutes at constant 150 Amps. All this done in a cold room.

Transfer time may vary according to the machine that you use. You should optimize the time for your own equipment. To do this, I recommend you to stain the gel after transferring and see if you have RNA left. If you do, next time transfer longer and repeat.

Hope it helps!

I agree with all the above answers. Capillary transfer is a reliable method which works quite well. However for a beginner electroblotting would work better with less effort and in short time efficiently. I attached a protocol for electroblotting agarsose gel on nylon membrane https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314723116657/litdocTechnicalBulletin121_20110830190234.pdf