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

Operation method

Labeling the 3' end of double-stranded DNA with the Klenow fragment of E. coli DNA polymerase I

Materials and Instruments

Restriction endonuclease E. coli DNA polymerase I Klenow fragment Template DNA
Ammonium acetate Ethanol dNTP solution
Sephadex G-50 column Water bath

Move

I. Materials

1. Buffers and solutions

Ammonium acetate (10 mol/L )

Ethanol

2. Enzyme and buffer

Suitable restriction endonucleases

E. coli DNA polymerase I Klenow fragments

3. Nucleic acids and oligonucleotides

dNTP solution containing 1 mmol/L of each of the appropriate unlabeled dNTPs

Template DNA (0.1-5 μg)

4. Radioactive complex

[ ^α-32P ] dNTP ( 10 mCi/ml, specific activity 800~3000 Ci/mmol)

5. Specialized equipment

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

Water bath pre-set to 75°C

ii. Methods

1. Digest up to 5 μg of template DNA with the desired restriction enzymes in 25-50 μl of appropriate restriction enzyme buffer.

2. To the completed restriction enzyme digestion reaction, add:

10 mCi/ml [ ^α-32P ] dNTP
(specific activity 800~3000 Ci/mmol) 2~50 μCi
Unlabeled dNTP to a final concentration of 100 μmol/L
Klenow fragment 1~5 units

The reaction was incubated at room temperature for 15 min.

3. Heat at 75°C for 10 min to terminate the reaction.

4. Separate radiolabeled DNA from undoped dNTP by chromatography on a Sephadex G-50 centrifugal column or by two rounds of ethanol precipitation in the presence of 2.5 mol/L ammonium acetate.

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