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Complete RNase T1 Digest
Posted on 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 ìl 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 ìl 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 ìl 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).
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 ìg 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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