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

Operation method

Preparation of radiolabeled DNA probes using the polymerase chain reaction

Materials and Instruments

Heat-resistant DNA polymerase Template DNA
Ammonium acetate Amplification buffer Chloroform Ethanol TE dCTP dNTP solution
Barrier Pipette Tips Microcentrifuge Tubes Forward Displacement Samplers Sephadex G-50 Centrifuge Columns Thermal Cyclers

Move

I. Materials

1. Buffers and solutions

Ammonium acetate (10 mol/L)

10X amplification buffer

Carrier for ethanol precipitation of radiolabeled probes

Chloroform

Ethanol

TE (pH 7.6)

2. Enzyme and buffer

Heat-resistant DNA polymerase (e.g., Taq DNA polymerase)

3. nucleic acids and oligonucleotides

dCTP ( 0.1 mmol/L)

dNTP solution containing 10 mmol/L each of dATP, dGTP, and dTTP

Forward primer (20 μmol/L) and reverse primer (20 μmol/L) (prepared in water)

Template DNA (2~10 ng)

4. Radioactive complex

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

5. Specialized equipment

Barrier tips for automated micro-sampler

Microcentrifuge tubes (0.5 ml, thin-walled tubes for PCR)

Positive displacement injector

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

Thermal cycler

II. Methods

1. Set up the following amplification/radiolabeling reaction system in a 0.5 ml thin-walled microcentrifuge tube:

10X Amplification Buffer 5.0 μl

10 mmol/L dNTP solution 1.0 μl

0.1 mmol/L dCTP 1.0 μl

20 μmol/L forward oligonucleotide primer 2.5 μl

20 μM reverse oligonucleotide primer 2.5 μl

Template DNA (2~10 ng or about 1 fmol) 5~10 μl

10 mCi/ml [ ^α-32P ] dCTP

(specific activity 3000 Ci/mmol) 5.0 μl

Water Add to 48 μl

Add 2.5 units of heat-resistant DNA polymerase to the reaction system. Tap the walls of the tube to mix the components.

2. If the thermal cycler does not have a heated lid, add a drop (50 μl) of light mineral oil or a bead of paraffin wax to the reaction mixture to prevent evaporation of the sample during repeated heating and cooling cycles. Place the microtube into the thermal cycler.

3. Amplify the sample by denaturation, denaturation and polymerization with the reaction times shown in the table below.



4. Remove the reaction tube from the thermal cycler and use a micropipette to aspirate as much mineral oil as possible from the upper layer of the reaction solution. Extract the reaction solution with 50 μl of chloroform to remove the remaining mineral oil. Centrifuge for 1 min at room temperature to separate the liquid phase from the organic phase.

5. Pipette off the upper aqueous phase, transfer to a clean microfuge tube, add carrier tRNA (10~100 μg) or glycogen (5 μg), precipitate the DNA with an equal volume of 4 mol/L ammonium acetate and 2.5 v/v of ethanol, and leave it at -20 ℃ for 1~2 h or -70 ℃ for 10~20 min. Centrifuge the precipitated DNA at maximal speed for 5~10 min at 4 ℃ to collect the precipitated DNA.

6. The DNA was dissolved in 20 μl of TE (pH 7.6) and chromatographed on a Sephadex G-75 centrifugal column to remove residual unincorporated dNTP and oligonucleotide primers.

7. Determine the radioactivity of a 1.0 μl volume of water outside the centrifuge column using a liquid scintillation counter. Store the remaining radiolabeled DNA at -20°C for backup.

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