Tuesday, October 21, 2003

Description
RNase T1 is an endonuclease that specifically cleaves 3' of G residues in RNA. This protocol uses RNase T1 for RNA mapping studies.

Procedure
1. Dry the RNA sample in either a vacuum centrifuge or a lyophilizer.

2. Resuspend the RNA in 25 l of Filter Buffer.

3. Add 2 to 20 Units of RNase T1.

4. Incubate at 37C for 30 min to 1 hour.

5. Add 325 l 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 l 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 90C prior to loading the sample onto a 15 to 20% Acrylamide plus 7 M Urea Gel (see protocol on Gel Electrophoresis of RNA).

Recipes
TBE Buffer (1X) pH 8.0
89 mM Tris
2 mM EDTA
89 mM Boric Acid


70% Ethanol 70% (w/v) Ethanol


SEVAG 24:1 Chloroform:Isoamyl alcohol


Phenol:SEVAG 1:1 Phenol:SEVAG


NET2 50 mM Tris, pH 7.4 using HCl
0.05% NP-40
150 mM NaCl


NET2 Mix 20 g carrier RNA
10 mM MgCl2
185 mM Sodium Acetate


Filter Buffer 0.2 M NaCl
1 mM EDTA
Filter sterilize
10 mM HEPES, pH 7.6 using NaOH


Loading Buffer Containing Urea 0.04% (w/v) Bromophenol Blue
in 1X TBE
10 M Urea (Saturated)



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