The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.

Operation method

Labeling the 3' protruding end of double-stranded DNA with [α-32P] 3'deoxyadenosine 5'-triphosphate or [α-32P] dideoxyATP ends

Materials and Instruments

Restriction endonuclease Calf thymus terminal transferase Template DNA
Ammonium acetate Ethanol Phenol Chloroform Terminal transferase buffer
Sephadex G-50 Centrifuge Columns

Move

I. Materials

1. Buffers and solutions

Ammonium acetate (10 mol/L )

Ethanol

Phenol: chloroform (1:1, V/V)

2. Enzyme and buffer

Appropriate restriction endonucleases

calf thymus terminal transferase

5X terminal transferase buffer

3. Nucleic acids and nucleotides

Template DNA ( 0.1~5 μg)

4. Radioactive compounds

[ ^α-32P ] 3' deoxyadenosine triphosphate (10 mCi/ml, specific activity 5000 Ci/mmol)

or

[ ^α-32P ] dideoxy ATP ( 10 mCi/ml, 3000 Ci/mmol)

5. Specialized equipment

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

II. Methods

1. Digest 0.1~5 μg of template DNA with appropriate restriction endonuclease.

2. Purify the DNA by phenol:chloroform extraction and standard ethanol precipitation.

3. Dissolve the digested DNA in 10 μl of 5X terminal transferase buffer and 34 μl of water.

4. Add 5 μl of 10 mCi/ml [ ^α-32P ] 3' deoxyadenosine 5'-triphosphate (5000 Ci/mmol) or [ ^α-32P ] dideoxy ATP (3000 Ci/mmol) and 1 μl of bovine thymus terminal transferase (~20 units).

5. Incubate the reaction for 1 h at 37°C.

6. Separate labeled DNA from unadulterated [ ^α-32P ] 3' deoxyadenosine 5'-triphosphate (or [ ^α-32P ] dideoxyATP) by centrifugal column chromatography on a Sephadex G-50 column or by two rounds of ethanol precipitation in the presence of 2.5 mol/L ammonium acetate.

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