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The Laboratory of Michael Chamberlin at the University of California, Berkeley
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1. Mix in a sterile microcentrifuge tube:
10 μl of End-labeled, single-stranded, radiolabeled oligonucleotide probe (dilute so that there are 10,000 to 50,000 cpm per reaction)
20 μl of Isolated Total RNA (or 20 μg carrier tRNA plus in vitro transcribed RNA)
10 μl of 3 M Sodium Acetate
Add ddH2O to a final reaction volume of 100 ul
2. Add 0.3 ml of 100% Ethanol and incubate for 10 min at -70°C.
3. Centrifuge in a microcentrifuge at maximum speed for 15 min. Decant the supernatant.
4. To the pellet add 1 ml of 70% Ethanol, mix well by inversion and incubate at -70°C for 5 min.
5. Centrifuge in a microcentrifuge at maximum speed for 15 min. Decant the supernatant.
6. Dry the pellet in a vacuum centrifuge.
7. Redissolve in 10 μl of Hybridization Buffer (see Hint #2).
8. Cove solution with 2 drops of mineral oil and centrifuge in a microcentrifuge at maximum speed for a couple of seconds to separate phases.
9. Denature the DNA by incubating for 5 min at 90°C.
10. Rapidly transfer to tubes to a 66°C water bath (do not allow samples to cool down during the transfer).
11. Allow the DNA to hybridize for 3 hours at 66°C.
12. Prepare S1 Digestion Mix in master tube (volumes are per reaction):
200 μl of S1 Digestion Buffer
2 Units of S1 Nuclease (see Hint #3)
13. Distribute 200 μl aliquots of the S1 Digestion Mix prepared in Step #12 into clean microcentrifuge tubes on ice.
14. Using a Pipette, remove hybridization "bubbles" from beneath the mineral oil and transfer to Digestion Mix tubes prepared in Step #13 (see Hint #4).
15. Mix well by vortexing and centrifuge in a microcentrifuge for a couple of seconds at maximum speed.
16. Incubate for 30 min at 37°C.
17. Add 600 μl of 100% Ethanol and incubate at -20°C for 1 hour.
18. Centrifuge in a microcentrifuge for 15 min at maximum speed.
19. Decant the supernatant and carefully redissolve the pellet in 10 μl of Loading Buffer.
20. Heat the solution for 2 min at 90°C then quickly chill in ice-water.
21. Electrophoresis according to protocol for DNA Electrophoresis.
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| Hybridization Buffer (5X) |
| 2M NaCl
0.2 M PIPES, pH 6.4
5 mM EDTA
Autoclave and store at -20°C
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| Polyacrylamide/Urea Gel |
| 200 μl of 10% (w/v) Ammonium Persulfate
10 ml ddH2O
15 g Urea
Filter and degas
3 ml 10X TBE
15 μl TEMED
6 ml 38% (w/v) Acrylamide:2% (w/v) Bisacrylamide (CAUTION! see Hint #1)
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| Loading Buffer |
| 0.04% Bromophenol Blue
0.04% Xylene Cyanol FF
80% (v/v) Deionized Formamide (CAUTION! see Hint #1).
1 mM EDTA
10 mM NaOH
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| S1 Nuclease Buffer |
| 1 mM Zinc Sulfate
0.25 M NaCl
50% (v/v) Glycerol
Store at -20°C
30 mM Sodium Acetate, pH 4.6
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| S1 Digestion Buffer |
| 5% (v/v) Glycerol
20 μg/ml sonicated, denatured Salmon Sperm DNA
1 mM Zinc Sulfate
0.25 M NaCl
Filter sterilize and store at -20°C
30 mM Sodium Acetate, pH 4.6
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Probe
Xylene Cyanol FF
Bromophenol Blue
S1 Nuclease
Tris Base
EDTA
Sodium Chloride
Mineral Oil
Zinc Sulfate
TEMED
Oligonucleotide
Ammonium Persulfate
Sodium Acetate
Boric Acid
Acrylamide
Urea
PIPES
Sodium Hydroxide
Glycerol
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. Slowly pipette the solution up and down. Briefly place the solution in a 37°C water bath. Repeat this cycle of pipetting and warming the solution until DNA is dissolved.
3. S1 Nuclease units are defined in this protocol as the amount of enzyme which hydrolyzes 1 μmole nucleotides in 30 min at 37°C. Different manufacturers use different units.
4. Do not worry about transferring the mineral oil into the S1 Digestion Mixture.
4. Electrophoresis the gel at approximately 12 to 20 mA.
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