Rapid isolation of yeast DNA

Summary

Yeast DNA prepared according to this protocol can be used as a template for PCR reactions. The shuttle plasmid, which replicates in both E. coli and Saccharomyces cerevisiae, can also be extracted from yeast and used for transformation of E. coli.

Operation method

Rapid isolation of yeast DNA

Principle

Yeast DNA prepared according to this protocol can be used as a template for PCR reactions. Shuttle plasmids that replicate in both E. coli and Saccharomyces cerevisiae can also be extracted from yeast and can be used to transform E. coli.

Materials and Instruments

Yeast Cells
Ethanol Phenol Chloroform Sodium Acetate STES Buffer TE
Acid-washed glass beads

Move

I. Materials

1. Buffers and solutions

Ethanol

Phenol: chloroform (1:1, V/V)

Sodium acetate (3 mol/L, pH 5.2)

STES buffer (0.2 mol/L Tris-Cl (pH 7.6), 0.5 mol/L NaCl, 0.1% (m/V) SDS, 0.01 mol/L EDTA, stored at room temperature)

TE ( pH 7.6)

2. Specialized equipment

Acid washed glass beads (0.4 mm)

3. Cells and tissues

Fresh agar plate cultured clones or liquid overnight cultured yeast cells

II. METHODS

1. Prepare yeast cells for lysis.

(1) Plate cultured yeast clones

Transfer one or several large, freshly cultured clones to a microcentrifuge tube with 50 μl of STES buffer using a sterile inoculation loop.

(2) Yeast in liquid culture

① Transfer 1.5 ml of overnight cultured yeast cells to a microcentrifuge tube.

② Centrifuge at maximum speed for 1 min at room temperature and collect the precipitated cells.

② Collect the precipitated cells by centrifugation at maximum speed for 1 min at room temperature. ③ Aspirate off the culture medium and resuspend the cells in 50 μl of STES buffer.

2. Add 50 μl of acid-washed glass beads to the yeast suspension. Add 20 μl TE (pH 7.6) to each tube.

3. Add 60 μl phenol/chloroform. Cover and shake for 1 min to mix the organic and aqueous phases.

4. Centrifuge at maximum speed for 5 min at room temperature.

5. Transfer the upper aqueous phase to a new centrifuge tube and precipitate with ethanol at 0°C for 15 min.

6. Centrifuge at maximum speed for 10 min at 4°C and collect the nucleic acid precipitate.

7. Aspirate off the supernatant and wash the precipitate with 100 μl of 70% ethanol in water. Centrifuge at maximum speed for 1 min at room temperature.

8. Aspirate the supernatant. Dry the DNA precipitate in air for 15 min. dissolve the precipitate in 40 μl TE (pH 7.6).

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