Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia
We have developed a quick and low-cost genomic DNA extraction protocol from yeast cells for PCR-based applications. This method does not require any enzymes, hazardous chemicals, or extreme temperatures, and is especially powerful for simultaneous analysis of a large number of samples. DNA can be efficiently extracted from different yeast species (Kluyveromyces lactis, Hansenula polymorpha, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris, and Saccharomyces cerevisiae). The protocol involves lysis of yeast colonies or cells from liquid culture in a lithium acetate (LiOAc)–SDS solution and subsequent precipitation of DNA with ethanol. Approximately 100 nanograms of total genomic DNA can be extracted from 1×107 cells. DNA extracted by this method is suitable for a variety of PCR-based applications (including colony PCR, real-time qPCR, and DNA sequencing) for amplification of DNA fragments of ≤3500 bp.
1.Pick one yeast colony from the plate or spin down 100–200 µL liquid yeast culture (OD600=0.4). Suspend cells in 100 µL 200 mM LiOAc, 1% SDS solution.
2. Incubate for 5 min at 70°C.
3. Add 300 µL 96–100% ethanol, then vortex.
4. Spin down DNA and cell debris at 15,000× g for 3 min.
5. Wash pellet with 70% ethanol.
6. Dissolve pellet in 100 µL H2O or TE and spin down cell debris for 15 s at 15,000× g.
7. Use 1 µL supernatant for PCR.
0.2 M lithium acetate (LiOAc) 1% SDS solution
Ethanol, 96–100% and 70%.