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Plasmid Extract from Budding Yeast (Saccharomyces cerevisiae)
Published: Jul 20, 2018 DOI: 10.21769/BioProtoc.2931 Views: 10242
Edited by: Chao Jiang Reviewed by: Samantha E. R. Dundon
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Abstract
Plasmids are widely used tools in yeast research. In many cases, plasmid libraries are used in genetic screens or in yeast two hybrid screens. In such cases, it is necessary to extract plasmids carrying unknown genetic elements from positive clones that were isolated in the screen.
This is a simple protocol to extract plasmid DNA from budding yeast cultures (Robzyk and Kassir, 1992). The amount produced is small, but it is sufficient for PCR or for transformation into bacteria, where the plasmid can be amplified to provide sufficient amounts for downstream uses (e.g., restriction enzyme analysis, sequencing).
Plasmid extraction
Miniprep
Background
Materials and Reagents
- Pipette tips (1,000 μl, 200 μl, 20 μl)
- 15 ml test-tube or equivalent
- 1.7 ml tubes
- 0.5 ml PCR tubes
- Kimwipes (e.g., KCWW, Kimberly-Clark, catalog number: 34120 )
- Isolated yeast clone carrying the required plasmid
- Difco Yeast nitrogen base without amino acid (e.g., BD, catalog number: 291940 )
- Yeast Synthetic Drop-out medium supplements (Sigma-Aldrich)
Note: Choose drop-out supplement based on the required plasmid selection in yeast (e.g., Sigma-Aldrich, catalog number: Y1501 for drop-out without Uracil). - D-Glucose (e.g., Sigma-Aldrich)
- Sucrose (e.g., Sigma-Aldrich)
- Tris base (e.g., Sigma-Aldrich)
- 12.EDTA (e.g., Sigma-Aldrich)
- Triton X-100
- 100% ethanol
- 70% ethanol
- Ammonium acetate
- HCl 1 N
- NaOH 10 N
- Nuclease-free water
- Double distilled water
- Ice
- 0.5 mm glass beads (e.g., MP Biomedicals, catalog number: 116540449-1kg )
- Synthetic selective medium (see Recipes)
- STET buffer (see Recipes)
- 7.5 M ammonium acetate (see Recipes)
Equipment
- 1,000 μl, 200 μl, 20 μl pipettes
- Yeast incubator/shaker suitable for 15 ml tubes or equivalent
- Refrigerated centrifuge suitable for 1.7 ml tubes
- Vacuum aspirator (optional)
- Heating block set to 100 °C
- Vortex mixer (optional: with multi-tube head ) (e.g., VWR, catalog number: 10153-836 )
- Oven (> 160 °C)
- -20 °C freezer
- Glass beaker (use appropriate size to amount of glass beads; a 1 L beaker is sufficient for 1 kg beads)
Procedure
Category
Microbiology > Microbial biochemistry > DNA
Molecular Biology > DNA > DNA extraction
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