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

Summary

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.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/