Total RNA Extraction using RNA-Bee (Tel-Test Inc., # CS-104B)
1. Turn on the refrigerated centrifuge. The power button is at the back Left. “x G” can be
changed to RPM. Get ice. Prepare collection area for RNase-free collection.
2. Put plates immediately on ice after removing from incubators.
3. Aspirate media. Rinse cells with 2ml cold 1X DPBS (Cellgro, 21-031-CV), 3 times each.
4. After the last wash, aspirate off as much DPBS as possible by tilting plate backwards.
5. Add 3 ml RNA-Bee directly to culture dish; remove plates from ice and lyse cells by
pipetting up and down.
6. Transfer lysate to labeled 15 ml centrifuge tube.
7. Add 0.6 ml chloroform (0.2 ml chloroform per 1 ml of RNA-Bee). Cap the tube and
shake vigorously for 15-30 seconds. Store the sample on ice for 5 minutes.
8. Centrifuge the homogenate at 3000 rpm for 15 minutes at 4°C
9. Prepare clean, labeled 15 ml centrifuge tubes with 8 ml of isopropanol.
10. Following centrifugation, transfer aqueous phase (upper colorless layer ≈ 1 ml) to labeled
15 ml centrifuge tubes with isopropanol.
11. Store the samples at -20°C overnight or longer.
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12. Turn on the refrigerated centrifuge. Centrifuge samples at 3000 rpm for 15 minutes at
4°C. RNA precipitate forms a clear gel pellet at the bottom of the tube.
13. Remove the supernatant and wash the RNA pellet with 75% ethanol (prepare 75%
ethanol with DEPC-treated RNase-free water! use at least 3 ml - can use excess
ethanol), shaking or vortexing to dislodge the pellet from the side of the tube. Centrifuge
at 3000 rpm for 15 minutes at 4°C.
14. Discard supernatant, and repeat wash in 75% ethanol. Centrifuge at 3000 rpm for 15
minutes at 4°C.
15. Pour a 1% agarose gel using equipment in RNA cabinet (dedicated to RNA gels).
16. At the end of the procedure, briefly air-dry the RNA pellet (Centrifuge at 3000 rpm for 510 minutes at 4°C open tubes). It is important not to let the RNA pellet dry completely, as
this greatly decreases its solubility.
Updated 2/13/14 CM
17. Dissolve the pellet in 15 l DEPC-treated RNase-free water.
18. Load 1.5L of each sample on nanodrop to measure concentration and purity.
=260 nm nucleic acid maximum of absorbance
=280 nm protein maximum of absorbance
=230 nm phenol maximum of absorbance
19. Prepare 500ng of sample + 2 L of blue loading dye (6X) + DEPC water qs for 12L in
RNase-free tubes. Load onto gel. Run at 80V / 400mA for one hour. Look for 28S and
18S bands. (28S bands (4.8 kb) should be more intense than 18S bands (1.9 kb), and there
should be no smear under 28S bands.)
20. Store samples at -80°C.
Updated 2/13/14 CM