Northern Blotting (Howell Lab)

RNA isolation

Harvest cells when they are about 80-85% confluent. Stationary phase cells have decreased RNA yield. Wash cells with warm PBS and scrape them off in 7 ml cold GITC buffer. Pour GITC/cell mix into (14x95 mm) polyallomer tubes with 4 ml CsCl buffer. Balance and top off tubes with more GITC buffer. Spin tubes on the SW40Ti rotor for 20 hr at 32,000 rpm (18℃). Pour off liquid and keep tubes inverted because the RNA pellets on the bottom. (DNA can be recovered from the CsCl layer.) Cut off the tube bottoms (keep inverted) and resuspend the pellet in 100 µl 0.3 M NaOAc (pH 6) buffer. Transfer to a screw-cap 1.5 ml Eppi. Wash tube bottom twice more with 100 µl buffer each time. Add 750 µl 100% EtOH and let precipitate overnight at -70℃. Spin down at 4℃ for 12 min, dry in the TC hood, and resuspend with DEPC water.

RNA quantitation

Let resuspended RNA sit on ice for at least 30 min before quantitation. Pipet up and down several times before taking 2 µl into 998 µl water. Read at 260 nm. RNA concentration (µg/µl) = OD x 500 x 0.04 µg/µl = OD x 20

Formaldehyde gel

Boil 2 g agarose in 144 ml water (check weight to account for evaporation) Add 20 ml 10X MOPS and let cool on stir plate to about 56℃. Add 36 ml 37% formaldehyde. Pour into Hoefer appartatus when gel solution is below 50℃. Running buffer: dilute 100 ml 10X MOPS to one liter.

Sample preparation

To 0.5 ml Eppis, add: a) 15 µg RNA (approxiamately 4.5 µl) b) 14.5 µl sample buffer c) Adjust with DEPC water so that final volumes are equal d) Heat for 5 min at 68℃, then chill on ice for 5 min. e) Add 2 µl loading buffer and 1 µl 0.4 mg/ml EtBr. For the ladder lanes, use 3 µg of 1 kb ladder and treat the same way as with the samples.

Running the gel

For the Hoefer, run between 70-85 V for about 3 hr or to a predetermined length. Recirculate the buffer with a peristaltic pump. As the gel is running, cut membrane, paper towels, etc. At the end, take a picture (f8, 4s) of the gel using a fluorescent ruler.