TEMPLATE PREPARATION TEMPLATE PREPARATION
1. Add 0.5 to 1 polyp equivalents to a sterile Eppendorf tube, remove Hydra Medium.
2. Lyse tissue with 100 µl freshly prepared GuSCN buffer (see below). Rapidly mix tissue and buffer with pipette WITHOUT foaming solution. Lysis should be completed in fewer than 30 sec.
3. Add 10 µl 2M Na-acetate pH 4. Mix with pipette.
4. Add 100 µl water-saturated phenol. Vortex briefly.
5. Add 20 µl chloroform-isoamy lalcohol (49:1). Vortex (should be cloudy). Place on ice 15 min. If phase separation NOT visible add 10 µl chloroform-isoamyl alcohol. Vortex.
6. Centrifuge at 10,000 x g 20 min at 4 degrees C.
7. Transfer aqueous phase (upper) to fresh Eppendorf tube.
8. Add 1/10 vol 3M Na-acetate pH 5.2. Mix with pipette.
9. Add 1 volume of isopropanol. Store at -20 degrees C for 2 hrs to overnight.
10. Centrifuge at full speed for 20 min at 4 degrees C.
11. Decant supernatant, spin pellet briefly, remove all supernatant. Gently air-dry pellet by placing tube, open, in 65 degrees C waterbath for 10-15 min. (This step is very important - alcohol will prevent pellet from resuspending in next step BUT overdrying will also cause resuspension problems).
12. Resuspend pellet in 20 µl GuSCN buffer.
13. Add 1/10 vol 3M Na-acetate pH 5.2 and 1 vol isopropanol. Store at -20 degrees C for at least 2 hrs.
14. Centrifuge at full speed for 20 min at 4 degrees C, keep pellet.
15. Wash pellet 5 X with 70% EtOH. (Traces of GuSCN in RNA will inhibit reverse transcriptase.)
16. After last wash, decant supernatant, spin pellet briefly, remove all supernatant. Gently air-dry pellet by placing tube, open, in 65 degrees C waterbath for 10-15 min.
17. Resuspend pellet in 6.5 µl DEPC-treated water. Use immediately in RT reaction.
Solutions:
- all should be prepared RNase-free. Use unopened chemicals, DEPC-treated water and good technique.
GuSCN Stock:
4 M GuSCN 25 mM Na-citrate, pH 7.0 0.5% Sarcosyl GuSCN Buffer (working solution):
add 10 µl of 2-mercaptoethanol per ml GuSCN Stock - use fresh
REVERSE TRANSCRIPTION REACTION
- 6.5 µl RNA - 4 µl MgCl2 (25 mM stock) - 4 µl 5X RT buffer (supplied with enzyme) - 2 µl 10 mM dNTP's - 0.5 µl RNasin - 1 µl Random Hexamer Primers (200 ng/µl in DEPC-treated water) - 2 µl AMV reverse transcriptase (approximately 10 units)
Incubate at room temperature for 10 min then at 42 degrees C for 1 hr.
Stop reaction - incubate at 95 degrees C for 5 min, place on ice for 5 min, add 20 µl 2x stop buffer (20 µM EDTA).
Store at -20 degrees C
POLYMERASE CHAIN REACTION
use 0.5 to 1 µl of product per 100 µl PCR reaction
Click here
to return to the Methods page.Click here to return to the Cnidaria home page.