Preparation of genomic DNA from mouse tails or other small samples

Summary

This simple protocol is widely used in hundreds of laboratories for genotyping transgenic or knockout mice, and for extracting DNA from small amounts of cultured cells or blocks of tissue.Source: "A Guide to Molecular Cloning, Third Edition", translated by Peitang Huang et al.

Operation method

Preparation of genomic DNA from mouse tails or other small samples

Principle

This simple protocol is widely used in hundreds of laboratories for genotyping transgenic or knockout mice and for extracting DNA from small amounts of cultured cells or tissue blocks.

Materials and Instruments

Proteinase K Cultured cells Mouse tail or mouse tissue
Ethanol Isopropanol Phenol Chloroform Isoamyl alcohol Phosphate buffer solution SNET TE
Sorvall centrifuge and H1000B, SH-3000 Turning head Polypropylene tubing Vibration platform Vibration platform or vibration incubator Shepherd's hooks

Move

I. Materials

1. Buffers and solutions

Ethanol

Isopropyl alcohol

Phenol: chloroform: isoamyl alcohol (25:24:1 V/V)

Phosphate buffer solution

SNET (20 mmol/L Tris-Cl ( pH 8.0), 5 mmol/L EDTA ( pH 8.0), 400 mmol/L NaCl, 1% (m/V) SDS)

TE ( pH 8.0)

2. Enzyme and buffer

Protease K (20 mg/ml)

Sorvall centrifuge with H1000B, SH-3000 turntable (or other equivalent)

3. Specialized equipment

Polypropylene tubing (17 X 100 mm)

Oscillating platform at room temperature and 4°C

Oscillating platform or vibration incubator preset to 55°C

Shepherd's Hook

4. Cells and tissues

Cultured cells

Mouse tail or mouse tissue

II. METHODS

1. Prepare an appropriate amount of lysis buffer, i.e., add proteinase K to SNET to achieve a final concentration of 400 μg/ml Add lysis buffer to mouse tail or other tissues.



2. Place the tubes vertically on a shaking platform or in a shaking thermostat at 55°C overnight.

It is very important that the samples are well mixed during digestion. After overnight digestion, no tissue or rat tail should be visible and the buffer should be a gray emulsion.

3. Add equal volumes of phenol/chloroform/isoamyl alcohol, seal the tube, and place on a shaker for 30 minutes at room temperature.

4. separate the organic and aqueous phases by centrifugation. samples in 17x100 mm Falcon polypropylene tubes are centrifuged at 666 g for 5 min at room temperature, i.e., Sorvall H1000B turntable with rotating drum at 1800 r/min, or Sorvall SH3000 rotating drum at 1600 r/min. for smaller samples the samples are centrifuged at maximum speed for 5 min at room temperature in microcentrifuge tubes. For smaller samples, the sample is centrifuged in a microcentrifuge tube at room temperature for 5 min at maximum speed and the upper aqueous phase is transferred to a new Falcon tube or microcentrifuge tube.

5. Add an equal volume of isopropanol to precipitate the DNA. centrifuge the sample at 13,250 g (8000 r/min, Sorvall 3000 head or maximum speed in a microcentrifuge tube) for 15 min at 4°C to collect the precipitated DNA.

6. Carefully remove the isopropanol. Soak the DNA precipitate in 1 ml of 70% ethanol. If the precipitate is loose, centrifuge for another 5 min, remove the 70% ethanol and dry the precipitate in air at room temperature for about 15-20 min.

Do not allow the DNA precipitate to dry completely or it will be very difficult to dissolve.

7. Add 0.5 ml TE and shake gently overnight at 4°C to dissolve the nucleic acid precipitate.

8. Transfer the solution to a microcentrifuge tube and store at room temperature.

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