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.
