实验步骤

1. Protocol for Removing 5' Phosphate

1) Combine the following in a single RNase-free microfuge tube:

-- µl Nuclease-free Water (to make a final volume of 10 µl)

-- µl RNA (0.1Ï10 pmol RNA)

1 µl 10X Dephosphorylation Buffer (0.5 M Tris, pH 8.5, 1 mM EDTA, pH 8)

1 µl Calf Intestinal Phosphatase (CIP; 0.1 U/µl)

2) Incubate 1 hr at 37°C.

Remove the Calf Intestine Alkaline Phosphatase by use of the Phosphatase Removal Reagent (available in the KinaseMax™ Kit) or by the following protocol:

3) To the above reaction, add:

125 µl water

15 µl 5.0 M ammonium acetate or 3.0 M sodium acetate

150 µl phenol/chloroform

Phenol extract by spinning the tube at room temperature in a microcentrifuge at its highest speed for 5 min. Transfer the aqueous (top) phase to a clean tube.

4) Ethanol precipitate by adding 450 µl 100% ethanol, vortex, and placing at -20°C or -80°C for 15 min. Spin the mixture at the highest speed setting in a microcentrifuge at 4°C for 15 min. Carefully withdraw the supernatant and discard. Wash the pellet once with ice cold 70% ethanol. Allow the remaining liquid to evaporate.

5) The pellet is now ready to be resuspended by addition of the individual components of the kinase reaction, below.

2. Protocol for 5' End Labeling RNA

1) Combine the following in a single RNase-free microfuge tube:

-- µl nuclease-free water (to make a final volume of 20 µl)

-- µl RNA (0.1 to 100 pmol RNA)

25 pmol [gamma-32P]ATP (7000 Ci/mmol, 150 mCi/ml)

2 µl 10X Kinase Buffer (500 mM Tris, pH 7.5, 100 mM MgCl2, 50 mM DTT)

1 µl T4 Polynucleotide Kinase (10 U/ml)

2) Incubate at 37°C for 1 hr.

3) (optional) Stop the reaction by adding EDTA to 1 mM, and then heating to 95¬C for 2 minutes.

4) (optional) Purify the reaction products. If it is important to remove free nucleotides, purification by spin-column chromatography (e.g., using Ambion­s NucAway Spin Columns) or denaturing polyacrylamide electrophoresis is recommended.