This protocol removes the 5' phosphate of a nucleic acid by phosphatase, and then re-adds the phosphate to the nucleic acid in a radiolabeled form catalyzed by T4 phage polynucleotide kinase, a technique widely used for 32P-labeled probes. This experiment is from "Molecular Cloning Laboratory Guide, Third Edition" translated by Huang Peitang et al.

Operation method

Phosphorylation of DNA molecules containing a 5' prominent hydroxyl terminus

Principle

This protocol removes the 5' phosphate of nucleic acids with phosphatase, and then re-adds the phosphate to the nucleic acids in a radiolabeled form catalyzed by T4 phage polynucleotide kinase, a technique widely used for 32P-labeled probes.

Materials and Instruments

T4 Phage polynucleotide kinase DNA
Ammonium Acetate EDTA Ethanol T4 Phage Polynucleotide Kinase Buffer
Liquid scintillation counter Sephadex G-50 centrifugal column Sephadex G-50 column

Move

I. Materials

1. Buffers and solutions

Ammonium acetate (10 mol/L)

EDTA ( 0.5 mol/L, pH 8.0 )

Ethanol

2. Enzyme and buffer

T4 phage polynucleotide kinase

10X T4 Phage Polynucleotide Kinase Buffer

3. Nucleic acids and oligonucleotides

DNA (10~50 pmol)

4. Radioactive complexes

[ ^γ-32P ] ATP ( 10 mCi/ml, specific activity 3000-7000 Ci/mmol)

5. Specialized equipment

Liquid scintillation counter for measurement of ^32P by Cherenkov radiation

Sephadex G-50 centrifuge column, equilibrated with TE (pH 7.6)

or

Sephadex G-50 column (1 ml) equilibrated with TE (pH 7.6)

ii. Methods

1. Mix the following reagents in a microcentrifuge tube:

Dephosphorylated DNA 10~50 pmol

10X T4 Phage Polynucleotide Kinase Buffer 5 μl

10 mCi/ml [ ^γ-32P ] ATP

( Specific activity of 3000~7000 Ci/mmol) 50 pmol

T4 phage polynucleotide kinase 10 units

Water Add to 50 μl

Incubate the reaction at 37℃ for 1 h.

2. Add 2 μl of 0.5 mol/L EDTA (pH 8.0) to terminate the reaction. The total radioactivity in the reaction mixture was measured by Cherenkov counting in a liquid scintillation counter.

3. Separate the radiolabeled probe from the unadulterated dNTP by one of the following methods:

Chromatography on a Sephadex G-50 centrifugal column.

or

conventional size exclusion chromatography on a 1 ml Sephadex G-50 column (equilibrated with TE)

or

Two rounds of selective precipitation of radiolabeled DNA with ammonium acetate and ethanol

4. The amount of radioactivity in the probe product is determined by Cherenkov counting, and the efficiency of transfer of the radiolabel to the 5' end is calculated by dividing the amount of radioactivity in the probe by the total amount of radioactivity in the reaction mixture.

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