| Contributor: |
The Laboratory of Donald Rio at the University of California, Berkeley
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| RNase T1 is an endonuclease that specifically cleaves 3' of G residues in RNA. This protocol uses RNase T1 for RNA mapping studies. |
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1. Dry the RNA sample in either a vacuum centrifuge or a lyophilizer.
2. Resuspend the RNA in 25 μl of Filter Buffer.
3. Add 2 to 20 Units of RNase T1.
4. Incubate at 37°C for 30 min to 1 hour.
5. Add 325 μl of NET2 Mix.
6. Add an equal volume of Phenol:SEVAG, mix well by inversion, centrifuge in a microcentrifuge at maximum speed to separate the phases, and save the aqueous phase (upper phase).
7. To the aqueous phase, add an equal volume of SEVAG, mix well by inversion, centrifuge in a microcentrifuge at maximum speed to separate the phases, and save the aqueous phase (upper phase).
8. To the aqueous phase add 750 μl of 100% Ethanol and mix well to precipitate the RNA.
9. Centrifuge in a microcentrifuge at maximum speed for 15 min to pellet the RNA and discard the supernatant.
10. To the pellet add an equal volume of 70% Ethanol. Mix well by inversion, centrifuge to pellet the RNA and discard the supernatant.
11. Invert the tubes to allow the pellet to air dry for approximately 5 minutes.
12. Resuspend the RNA in ddH2O.
13. Dry down the pellet in a either a vacuum centrifuge or a lyophilizer.
14. Resuspend the RNA in Loading Buffer Containing Urea.
15. Incubate for 90 sec at 90°C prior to loading the sample onto a 15 to 20% Acrylamide plus 7 M Urea Gel (see protocol on Gel Electrophoresis of RNA).
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| NET2 |
| 50 mM Tris, pH 7.4 using HCl
0.05% NP-40
150 mM NaCl
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| NET2 Mix |
| 20 μg carrier RNA
10 mM MgCl2
185 mM Sodium Acetate
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| Filter Buffer |
| 0.2 M NaCl
1 mM EDTA
Filter sterilize
10 mM HEPES, pH 7.6 using NaOH
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| Loading Buffer Containing Urea |
| 0.04% (w/v) Bromophenol Blue
in 1X TBE
10 M Urea (Saturated)
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| TBE Buffer (1X) |
| pH 8.0
89 mM Tris
2 mM EDTA
89 mM Boric Acid
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| 70% Ethanol |
| 70% (w/v) Ethanol
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| SEVAG |
| 24:1 Chloroform:Isoamyl alcohol
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| Phenol:SEVAG |
| 1:1 Phenol:SEVAG
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Boric Acid
Ethanol
Acrylamide
Sodium Chloride
Sodium Acetate
Potassium Hydroxide
Tris
RNase, T1
Bromophenol Blue
Magnesium Chloride
Isoamyl Alcohol
Chloroform
Phenol
Hydrochloric Acid
HEPES
Urea
NP-40
EDTA
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No hints are associated with this bioProtocol
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