protocol

Introduction

T4 Polynucleotide Kinase (PNK) catalyzes the transfer of the gamma-phosphate of ATP to the 5'-hydroxyl termini of DNA or RNA. This phosphate transfer is commonly referred to as a kinase or phosphorylation reaction ([gamma-32P]ATP is often used in the reaction). Nucleic acids with a 5'-hydroxyl (OH) group can be added directly to a kinase reaction.

Nucleic acids with a 5'-phosphate should be dephosphorylated (e.g., with Calf Intestinal Phosphatase) prior to labeling with PNK and [gamma-32P]ATP. Inactivating the Calf Intestinal Phosphatase (CIP) after the dephosphorylation reaction typically requires phenol extraction and ethanol precipitation, although the KinaseMaxØ 5' End Labeling Kit includes a novel Phosphatase Removal Reagent for quick and complete removal of CIP without phenol extraction or ethanol precipitation. After the dephosphorylation step, the reagent is added to the reaction and incubated at room temperature for 2 minutes. The tube is then spun for a minute in a microfuge and the supernatant transferred to a new tube for the kinase reaction.

The below protocol is for 5' end labeling RNA with PNK. The labeled RNA can be used as a probe or for RNA structure assessment, protein footprinting, and boundary determination experiments.

Protocol for Removing 5' Phosphate

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


    2. -- 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.
TOP

Protocol for 5' End Labeling RNA

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

    2. -- 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.

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