mec

Rapid isolation of yeast total DNA

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Extraction of Yeast Chromosomal DNA with Ethanol Precipitation

Overview

This protocol is similar to this protocol, but includes an ethanol precipitation step at the end. It has been used extensively and usually works very well.

PCR inhibition

At one point, PCR was not possible when undiluted DNA was used as the template, whereas PCR products were obtained from 1:10 dilutions. This may have been caused by oxidized phenol or another inhibitor carried over during extraction.

Reference

Hoffman, Charles S. 2001. “Preparation of Yeast DNA.” In Current Protocols in Molecular Biology. John Wiley & Sons, Inc. (pdf)

Applications

The DNA preparation described in the original protocol can be scaled up to prepare chromosomal DNA for:

  • Southern hybridization analysis
  • PCR amplification
  • Restriction digestion and ligation
  • Cloning of integrated plasmids

This procedure is significantly faster than many other protocols used to isolate high-molecular-weight DNA.

Although the DNA is subject to shearing during the procedure, the resulting DNA is of sufficiently high molecular weight to allow the detection of restriction fragments up to approximately 19 kb by Southern hybridization.

Materials

Reagents

  • TE buffer

  • Breaking Buffer

  • Phenol:chloroform

  • DNase-free RNase A: 1 mg/mL solution, or 20 mg/mL solution

  • 4 M ammonium acetate, or 10 M ammonium acetate

  • 99–100% ethanol

  • Sterile water

  • Glass beads, approximately 0.3 g per sample

Equipment

  • 15 mL polypropylene Falcon tubes
  • 1.5 or 2 mL microcentrifuge tubes
  • Tabletop centrifuge
  • Microcentrifuge
  • Vortex mixer
  • Incubator or heating block set to 37 °C
  • Microscope, optional

Procedure

1. Prepare the yeast cells

  1. Grow a 10 mL yeast culture in YPD overnight to stationary phase in a sterile 50 mL polypropylene Falcon tube.

  2. Centrifuge the culture for 1 minute at 5,000 rpm.

    Room-temperature or chilled centrifugation may be used.

  3. Pour off the supernatant and resuspend the cells in 0.5 mL sterile water.

  4. Transfer the resuspended cells to a microcentrifuge tube.

  5. Centrifuge for 5 seconds at room temperature.

  6. Pour off the supernatant.

    Note

    This water-washing step removes any remaining culture medium.

  7. Disrupt the pellet by vortexing briefly.

2. Break open the cells

  1. Resuspend the cells in 200 µL Breaking Buffer.

  2. Add approximately 0.3 g glass beads, corresponding to approximately 200 µL by volume.

  3. Add 200 µL phenol:chloroform.

  4. Vortex at maximum speed for 3 minutes.

    Phenol:chloroform phase lower phase in the stock container. The upper phase is the aqueous buffer.

    The phenol:chloroform is the

    Optimising cell breakage

    The amount of vortexing required may vary depending on the vortex mixer.

    Determine by microscopic examination the minimum vortexing time required to break approximately 80–90% of the cells.

    When using a multi-tube vortex rather than a single-tube vortex, the vortexing time may need to be increased to 4–5 minutes.

    Longer vortexing may improve cell breakage but can also increase DNA shearing.

  5. Add 200 µL TE buffer and vortex briefly.

  6. Microcentrifuge for 5 minutes at high speed and room temperature.

  7. Transfer the upper aqueous phase to a clean microcentrifuge tube.

    Avoid transferring material from the interface or lower organic phase.

  8. Add 1 mL 99% ethanol.

  9. Mix by inversion.

  10. Microcentrifuge for 3 minutes at high speed and room temperature.

  11. Remove the supernatant.

  12. Resuspend the pellet in 400 µL TE buffer.

3. Degrade RNA contaminants and recover the DNA

Note

RNase treatment is optional but recommended when RNA-free DNA is required.

  1. Add one of the following:

    • 30 µL of 1 mg/mL DNase-free RNase A, or
    • 1.5 µL of 20 mg/mL DNase-free RNase A

    Both alternatives provide approximately 0.03 mg RNase A.

  2. Mix and incubate for 5 minutes at 37 °C.

  3. Add one of the following:

    • 10 µL of 4 M ammonium acetate, or
    • 4 µL of 10 M ammonium acetate

  4. Add 1 mL 100% ethanol.

  5. Mix by inversion.

  6. Microcentrifuge for 3 minutes at high speed and room temperature.

  7. Discard the supernatant carefully.

  8. Allow the DNA pellet to dry.

    Do not overdry the pellet, as this may make it difficult to resuspend.

  9. Resuspend the DNA in 100–200 µL TE buffer.

Expected yield

A yield of approximately 20 µg chromosomal DNA should be obtained.

The DNA can be used for:

  • Restriction digestion
  • PCR amplification
  • Southern blot analysis

Southern blotting

For best results:

  • Use 5 µL DNA, corresponding to approximately 1 µg DNA
  • Perform the digestion in a total volume of 20 µL

PCR

Use:

  • 2 µL DNA
  • In a total PCR volume of 50 µL

Tip

When PCR inhibition is suspected, test a 1:10 dilution of the extracted DNA.

Breaking Buffer

Composition

ComponentFinal concentration
Triton X-1002% v/v
Sodium dodecyl sulfate (SDS)1% v/v
NaCl100 mM
Tris-Cl, pH 8.010 mM
EDTA, pH 8.01 mM

Storage

Store at room temperature for up to 1 year.

Graph View

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