In the forward reaction of T4 polynucleotide kinase, DNA substrates with flat ends, 5' concave ends or intramolecular cuts are less efficient than molecular labeling with 5' protruding ends. The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.

Operation method

Phosphorylation of dephosphorylated flat or 5' concave end DNA molecules

Principle

DNA substrates with flat ends, 5' concave ends, or intramolecular incisions are less efficient than molecular labeling of 5' protruding ends in the forward reaction of T4 polynucleotide kinase.

Materials and Instruments

T4 Phage polynucleotide kinase
Ammonium Acetate EDTA Ethanol Imidazole Buffer Polyethylene Glycol
Liquid Scintillation Counters Sephadex G-50 Centrifugal Columns Sephadex G-50 Columns

Move

I. Methodology

1. Buffers and solutions

Ammonium acetate (10 mol/L)

EDTA ( 0.5 mol/L, pH 8.0)

Ethanol

10X imidazole buffer (500 mmol/L imidazole hydrochloride (pH 6.4), 180 mmol/L MgCl₂, 50 mmol/L DTT, 1 mmol/L spermidine hydrochloride, 1 mmol/L EDTA (pH 8.0))

Water formulated polyethylene glycol (24% m/V PEG 8000)

2. Enzyme and buffer

T4 phage polynucleotide kinase

3. Nucleic acids and oligosiderophores

DNA (10 to 50 pmol, volume ≤ 11 μl)

4. Radioactive complexes

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

5. Specialized equipment

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

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

or

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

ii. Methods

1. In a microcentrifuge tube, mix in the following order:

Dephosphorylated DNA 10~50 pmol

10X Imidazole Buffer 4 μl

Water Add to 15 μl

24% (m/V) PEG 10 μl

2. Add 40 pmol of [ ^γ-32P ] ATP (10 mCi/ml; specific activity 3000 Ci/mmol) and add water until the final volume is 40 μl.

3. Add 40 units of T4 phage polynucleotide kinase to the reaction. Flick the wall of the tube to mix the reagents and incubate the reaction at 37°C for 30 min.

4. Add 2 μl of 0.5 mol/L EDTA (pH 8.0) to terminate the reaction. Determine the total activity of the reaction mixture by Cherenkov counting on a liquid scintillation counter.

5. Separate the radiolabeled probe from the undoped dNTP by the following method:

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

6. Measure the activity of the probe by Cherenkov counting. Calculate the efficiency of transfer of the radiolabel to the 5' end by dividing the activity of the probe by the total activity in the reaction mixture.

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