Quantification of mRNA in Neuronal Tissue by Northern Analysis - Drugs of Abuse: Neurological Reviews and Protocols - page 165

Fig. 1. Assembly of the electroblotter. Put the fi ber pad provided with the transfer
unit into a tray with gel transfer buffer (1X TAE), and remove all air bubbles by gently
pressing on the fi ber pad with a gloved hand. Add a piece of strong fi lter paper and
let wet thoroughly. Add the precut nylon membrane and let wet. Put the gel on top
followed by a second fi ber pad. Make sure there are no air bubbles between any
layers of the sandwich. Close the cassette and slide into the electroblotter. The red
powercord (leading to the anode) needs to be on the side of the membrane, and the
black powercord (leading to the cathode) on the side of the gel.

a gel box with a buffer circulation system or stir the gel on a magnetic stirrer
during the run.

2. In each lane, load between 5 and 10 g of total RNA, or the entire sample in

the case of RNA from neuronal cultures. Load equal amounts of RNA within
an experiment.

µ

3. Run samples at 100-150 V for 90 min to 3 h (depending on the size of the
RNA of interest and separation needed). If you do not have a buffer circulation
system or cannot stir the gel during the run, reduce to 80-90 V and increase
running time.

4. Turn off the power supply, disconnect the cables, remove the lid, and take out
the gel.

5. Prepare the electroblotting tank (vertical buffer tank) with 1X TAE buffer, and
cut the nylon membrane and fi lter paper to size of gel ( see Fig. 1 ).

6. Assemble the electroblotting sandwich in a tray with transfer buffer as shown
in Fig. 1 .

7. Electroblot RNA onto nylon membrane ( Fig. 1 ) at constant current of 1.2 amp
for 2 h. RNA moves toward the anode (positive electrode).