From Bottom Up

3 4 cm paper towel, cut 1 2 cm wider than gel.

3 sheets Chromatography Paper, Dry. Cut slightly larger than gel.
2 sheets Chromatography Paper, wet in transfer buffer. Cut slightly larger than gel.
Membrane
Gel
3 sheets Chromatography Paper, wet in transfer buffer. Cut slightly larger than gel.
Bridge. 2 or 3 sheets Chromatography paper, wet in Transfer buffer, for Bridge.
Cover (us a gel tray) with 200 g weight added on top.
Reservoir tray to hold 35 40 mL Transfer Buffer.
Support for Reservoir tray.
Put a long pipet tube below the Bridge and next to gel so that the Bridge does not touch
the sides of the gel or chromatography paper.

Summary of Northern gel procedures:

1. Use RNase Zap on your gloves.
2. Prepare Running Buffer and Gel.
2.1. Dilute 10X Running Buffer to 1X (100 mL 10X and 900 mL Millipore water)
2.2. Prepare Gel. 100 mL 1X Running Buffer and 1 gram LE Agarose.
2.3. Set up Electrophoresis chamber.
3. Prepare RNA
3.1. Mix 10 uL RNA and 10uL Glyoxal Load Dye in heat tolerant 1 mL tube that comes with
the kit.
3.2. Incubate 30 min. at 50 C in the heat block
4. Put gel in electrophoresis chamber and add Transfer Buffer.
4.1. Pipet RNA samples into gel.
4.2. Run electrophoresis at 100 125 V.
4.3. When done, examine gel in UV box and photograph.
5. Transfer to membrane.
5.1. Use nylon membrane.
5.2. Place gel on transfer structure (see directions for assembling transfer structure).
5.3. Transfer requires 1.5 2 hours.
5.4. When transfer is complete, rinse membrane in running buffer (can use buffer remaining
in electrophoresis chamber.
5.5. Crosslink the membrane (see separate directions).
5.6. Store membrane in freezer
6. Prehybridization.
6.1. Preheat ULTRAhyb to 68 C.
6.2. Place membrane in a roller tube
6.3. Add about 70 mL ULTRAhyb to roller tube
6.4. Incubate in oven at appropriate temperature for 30 minutes.
6.5. Add Probe. See BrightStar Psoralen-Biotin Kit for directions on preparing probe
(Part D, Page 9). Hybridize 2 hours to overnight.
7. For wash instructions go to BrightStar Bio Detect Kit procedures.

Schematic Protocol of Northern Blot assay:

1.
2.
3.
4.
5.
6.
7.
8.

Make Probes for Hybridization (biotin-labeled probes).

Calculate RNA amount used in the experiment (adjust to 10 ug).
Denature RNA for 30 min, at 50oC with Glyoxal dye.
Cast 1.2 % agarose Gel. Run RNA samples on a gel (take a picture for QC).
Transfer the RNA on the Membrane (~2h).
UV-crosslinked Membrane.
Hybridization: Preheat ULTRAhyb (Ambion) to 68 oC.
Hybridize at 42-68 oC for O/N. Wash. Develop. Scan on Kodak scanner.