Using oligonucleotides as primers, DNA polymerase can initiate DNA synthesis along a single-stranded template (Gotilian 1969). If the sequences of the oligonucleotides are not homogeneous, they can hybridize to the template at multiple sites on the template. Thus the complement of each nucleotide on the template (except those at the 5' end) will be incorporated into the product at the same frequency. The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition" translated by Huang Peitang et al.

Operation method

Random priming method (labeling DNA radioactivity using oligonucleotide extension)

Principle

Using oligonucleotides as primers, DNA polymerase can initiate DNA synthesis along a single-stranded template (Gotilian 1969). If the sequences of the oligonucleotides are not homogeneous, they can hybridize to the template at multiple sites on the template. Thus the complement of each nucleotide on the template (except those at the 5' end) will be incorporated into the product at the same frequency.

Materials and Instruments

E. coli DNA Polymerase I Klenow Fragment Random Deoxynucleotide Primer Template DNA
Ammonia acetate Ethanol NA termination Storage buffer Random primer buffer dNTP solution
Basic agarose gel or denaturing polyacrylamide gel Boiling water bath or heated plate Microcentrifuge tubes SephadexG-50 columns

Move

I. Materials

1. Buffers and solutions

Ammonia acetate (10 mol/L)

Ethanol

NA termination/storage buffer (50 mmol/L Tris-Cl (pH 7.5), 50 mmol/L NaCl, 5 mmol/L EDTA (pH 8.0), 0.5% (m/V) SDS)

5X random priming buffer (250 mmol/L Tris (pH 8.0), 25 mmol/L MgCl₂, 100 mmol/L NaCl, 10 mmol/L di-Threitol (DTT), 1 mol/L HEPES (adjusted to pH 6.6 with 4 mol/L NaOH), 1 mol/L DTT was stored at -20°C, diluted with water and discarded after use. 1 mol/L DTT was stored at -20°C, diluted with water before use, and the diluted DTT was discarded after use.)

2. Enzyme and buffer

E. coli DNA polymerase Ⅰ Klenow fragments

3. gel

Basic agarose gel or denaturing polyacrylamide gel

4. Nucleic acids and oligonucleotides

Contains three unlabeled dNTP solutions (5 mmol/L each)

Random deoxynucleotide primers of six or seven bases in length (125 ng/μl TE, pH 7.6)

Template DNA ( 5~25 ng/μl TE, pH 7.6)

5. Radiocomplexes

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

6. Specialized equipment

Boiling water bath or heating plate preheated to 95°C

Microcentrifuge tube (0.5 ml )

SephadexG-50 column, equilibrated with TE (pH 7.6)

II.

1. Add template DNA (25 ng) dissolved in 30 μl of water and 1 μl of random deoxynucleotide primer (~125 ng) to a 0.5 ml microcentrifuge tube. Cap the microcentrifuge tube tightly and place it in a boiling water bath for 2 min.

2. Transfer the microcentrifuge tube to ice for 1 min, centrifuge at 4°C for 10 s to allow the primer-template mixture to settle to the bottom of the tube, and place the microcentrifuge tube back on ice.

3. Add to the mixture of primer and template:

5 mmol/L dNTP solution 1 μl

5X random primer buffer 10 μl

10 mCi/ml [ ^α-32P ] dNTP 5 μl

( Specific activity 3000 Ci/mmol)

Water Add to 50 μl

4. Add 5 units (~1 μl) of Klenow Fragment and flick the walls of the tube to mix the components. Centrifuge at maximum speed for 1-2 s to allow all liquid to settle to the bottom of the tube. React the reaction mixture for 60 min at room temperature.

5. Add 10 μl of NA Termination/Storage Buffer to the reaction solution and proceed as necessary.

Store the radiolabeled probe at -20°C for hybridization.

OR

Separate radiolabeled probe from unbound dNTP by centrifugal column chromatography or selectively precipitate radiolabeled DNA with ammonium acetate and ethanol; this step may be omitted if >50% of the radiolabeled dNTP has been incorporated into the reaction.

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