Protocol PolyA Site Cleavage in HeLa Cell Nuclear Extract

1. Reagents

HeLa cell nuclear extract (prepared by the method of Ruegsegger et al, 1996): dialyzed (~24 mg protein/ml dialyzed into buffer B) or undialyzed (~29 mg protein/ml)

a-32P-Labeled pre-mRNA substrate, —25 fmol//xl (—4 X 103 Cerenkov cpm/fmol), resuspended in H20 Buffer A [20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 7.9), 0.2 mMEDTA, 10% glycerol, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 mM dithiothreitol (DDT)], required if undialyzed extract is used Buffer B [100 mM KCl, 20 mM HEPES (pH 7.9), 0.2 mMEDTA, 10% glycerol, 0.1 mM PMSF, 0.5 mMDTT], required if dialyzed extract is used 3' dATP (12.5 mM) Polyvinyl alcohol (PVA) (10%) tRNA (0.5 mg/ml) tRNA (10 mg/ml)

ETS buffer [10 mMEDTA, 10 mMTris (pH 7.8), 0.5% SDS] Proteinase K (20 mg/ml) Sodium acetate (pH 6.1) (3 M)

Phenol, phenol/chloroform/isoamyl alcohol (25 : 24 :1) 95% Ethanol, 75% ethanol

7 M Urea Polyacrylamide gel, 90% formamide loading dye

2. Methods

1. Reactions (25 /xl) are set up on ice by the addition of individual components in the following order:

12 /U.1 Buffer B and 5 /xl dialyzed nuclear extract or 14.5 /xl buffer A

and 2.5 /xl undialyzed nuclear extract 1 /xl 32P-Labeled pre-mRNA substrate

2. Incubate the reactions at 30°C for 30 to 90 mins.

3. Add 175 /xl ETS buffer and 5 /xl proteinase K. Incubate the reactions for 10 min at 37°C.

4. Extract the reaction with 200 /xl phenol. Remove the aqueous phase and add 25 /xl of 3 M sodium acetate and 1 /xl tRNA (10 mg/ml). Reextract the aqueous phase with 200 /xl phenol/chloroform/isoamyl alcohol.

5. Precipitate the RNA with 2.5 volumes of 95% ethanol, wash the pellet with 75% ethanol, and dry the pellet. Resuspend the pellet in 5 /xl loading dye, boil for 1 min, and quickly chill in an ice-water bath. Load onto a denaturing Polyacrylamide gel (prerun for 30 min).

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