Labeling the 3' end of double-stranded DNA with T4 phage DNA polymerase
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 T4 phage DNA polymerase
Materials and Instruments
Restriction endonuclease T4 phage DNA polymerase Template DNA Move I. Materials For more product details, please visit Aladdin Scientific website.
Ammonia acetate Ethanol Phenol Chloroform T4 phage DNA polymerase buffer dNTP solution
Sephadex G-50 column Water bath
1. Buffers and solutions
Ammonia acetate (10 mol/L)
Ethanol
Phenol: chloroform (1:1, V/V)
2. Enzyme and buffer
Appropriate restriction endonucleases
T4 phage DNA polymerase
10X T4 Phage DNA Polymerase Buffer
3. Nucleic acids and oligonucleotides
Contains three labeled dNTP solutions at 2 mmol/L each.
A solution containing one unlabeled dNTP at a concentration of 2 mmol/L
Template DNA (0.1~5 μg)
4. Radiocomplexes
[ 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 preset to 70 °C
II.
1. Digest 0.1-5.0 μg of template DNA with a restriction enzyme that produces a flat or 3' protruding end.
2. Purify the DNA by phenol/chloroform extraction and ethanol precipitation in the presence of 2.5 mol/L ammonium acetate.
3. Dissolve the DNA precipitate in the presence of 2.5 mol/L ammonium acetate:
10X T4 Phage DNA Polymerase Buffer 2 μl
2 mmol/L three unlabeled dNTP solutions 1 μl
10 mCi/ml [ α-32P ] dNTP
( 800~3000 Ci/mmol) 1 μl
T4 phage DNA polymerase (2.5 units/μl) 1 μl
Water Add to 20 μl
Incubate at 37℃ for 5 min.
4. Add 1 μl of 2 mmol/L unlabeled fourth dNTP solution and continue to incubate for 10 min.
5. Heat at 70℃ for 5 min to terminate the reaction.
6. Separate the labeled DNA from the 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.