RNA Preparation and Northern blotting Protocol

Powers Lab UC Davis 9/01

Note: Protocol is designed for 50 ml of cells grown to mid log (OD600 ~ 0.5-0.7). This will provide enough RNA for several Northern blots. In general, however, this is not a sufficient amount to continue with Poly A mRNA isolation, where a minimum of 250 ml of culture is recommended.

Following experiment, pellet cells in 50 ml plastic conical centrifuge tubes
Discard media and quick-freeze cells in liquid nitrogen. Store at -80°C until ready for RNA prep.

Phenol Extractions Using Phase-lock tubes

Thaw 50 ml tubes on ice
Add 10 ml of AE Buffer to each tube
[AE Buffer= 50 mM NaOAc (pH 5.5), 10 mM EDTA]
Add 1 ml 10% SDS and 10 ml water-saturated phenol to each tube
Vortex 1 minute, place in 65°C bath for 10 minutes, revortex 30 seconds
Spin tubes in clinical centrifuge at 4,000 rpm for 10 minutes
[Also spin phase lock tubes at the same time (Eppendorf, 50 ml Phase Lock Gel, Heavy)]
Pour contents of tube into pre-spun phase lock tube (aqueous and organic phases will mix-this is okay), discard pellet
Add 10 ml chloroform. Mix by inverting gently several times (Do Not Vortex).
Spin tubes in clinical centrifuge 10 minutes
Pour aqueous contents of tube into new 50 ml falcon tube, discard organic phase
Add 1/10 volume 3M NaOAc pH 5.5 (~1.2 ml)
Add equal volume of isopropanol (~13 ml); mix
Store -20°C for 2 hours to overnight.
Spin cells in SS-34 type rotor that will accept conical centrifuge tubes (or transfer contents to conventional centrifuge tubes) at 10K rpm for 20 minutes.
Discard supernatant and wash pellets with 10 ml 70% EtOH
Dry pellets under vacuum
Resuspend pellets in ~100 µl RNase free H2O (concentration should be in the range of 2-10 mg/ml)
Store RNA at -80°C

Northern blotting Protocol

Overview: the protocol is broken into 4 basic steps: 1. Outline of all equipment and reagents used, 2. Sample prep and running the gel, 3. Transfer of RNA to nylon membrane, and 4. Probing the blot.

Equipment and reagents used:

Gel Box: Model H4 Horizon Gel Electrophoresis Apparatus
Life Technologies Cat. No. 11025-012

Gel Combs: 1.0 mm thick 15 tooth comb
Life Technologies Cat. No. 11953-072
(note: I think this particular comb makes all of the
difference in the quality of the resulting bands on the blot)

Power supply: BioRad Powerpac 300 or equivalent

Sample denaturation mix
323 µl formamide
113 µl formaldehyde
65 µl 10X E Buffer
0.5 µl Ethidium Bromide

Duralon-UV membrane (Stratagene, Cat No. 420101)
Ready-to-go DNA labeling beads -dCTP (Amersham Pharmacia)
a-labeled dCTP High specific activity (> 4000 Ci/mmol)
Quick spin G-50 sephadex TE spin columns (Boehringer, Cat. No. 1523-023)

Formaldehyde-Agarose Gel Electrophoresis

The gel:
Combine 250 ml H2O and 5.25 grams agarose in 500 ml flask
Heat in microwave until agarose is dissolved
Add stir bar and place on stir plate in a fume hood

Combine 35 ml 10X E Buffer and 65 ml formaldehyde in a 100 ml glass graduated cylinder and mix

Add to agarose slowly and let cool until flask can be held with gloved hands

Pour into gel tray that comes with gel box (combs are inserted and ends are taped to prevent leaks). Let solidify ~30 minutes

Sample prep:
Combine:
15 µl Sample denaturation mix
6 µl RNA (10-20 µg RNA and H2O to volume)

Heat 15 minutes 55°C, then sit on ice for 2 minutes
Add 2 µl of Loading Mix

Gel running:
Set up gel in gel box and add 2 liters of 1X E Buffer
Load Samples
Run at 100 V for 3-5 hours