Aug 16, 2022

Public workspaceDNA Extraction from Yeast

This protocol is a draft, published without a DOI.
DNA Extraction from Yeast
  • Brian Teague1
  • 1University of Wisconsin - Stout
  • Yeast ORFans CURE
Brian Teague
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Protocol Citation: Brian Teague 2022. DNA Extraction from Yeast. protocols.io https://protocols.io/view/dna-extraction-from-yeast-cfagtibw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 15, 2022
Last Modified: August 16, 2022
Protocol Integer ID: 68648
Keywords: dna, extraction, saccharomyces, pcr, lioac, sds, dna extraction from yeast, genomic dna out of yeast cell, dna extraction, pcr template, genomic dna, yeast cell, yeast
Abstract
This is a "quick and dirty" way to get some genomic DNA out of yeast cells. Not pure enough for many things, but should be fine for a PCR template.

This protocol is adapted from
Citation
Blount BA, Driessen MR, Ellis T (2016). GC Preps: Fast and Easy Extraction of Stable Yeast Genomic DNA. Scientific reports.
https://doi.org/10.1038/srep26863
LINK
who in turn adapted it from
Citation
Lõoke M, Kristjuhan K, Kristjuhan A (2011). Extraction of genomic DNA from yeasts for PCR-based applications. BioTechniques.
https://doi.org/10.2144/000113672
LINK


Image Attribution
By gskx via Flickr. https://www.flickr.com/photos/gskx/89462961
Guidelines
The centrifugation steps all specify Centrifigation21000 x g . If your microcentrifuge doesn't go this high, spin at the fastest speed available.

Materials
Equipment
  • Dry bath at 72°C
  • Dry bath at 42°C (optional)

Materials
  • Sterile water
  • ReagentLithium Acetate DihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #L4158 solution, Concentration1 Molarity (M)
  • ReagentSodium dodecyl sulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #436143-25G solution, Concentration10 Mass / % volume
  • ReagentEthanol (100%, Molecular Biology Grade)Fisher ScientificCatalog #BP2818500
  • ReagentEthanol (100%, Molecular Biology Grade)Fisher ScientificCatalog #BP2818500 solution, Concentration70 % (v/v)
  • ReagentTE Buffer









Protocol materials
ReagentEthanol (100%, Molecular Biology Grade)Fisher ScientificCatalog #BP2818500
ReagentTE Buffer
ReagentLithium Acetate DihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #L4158
ReagentSodium dodecyl sulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #436143-25G
Troubleshooting
Safety warnings
Both lithium acetate and SDS are irritants, particularly in the eyes. Wear appropriate PPE, including safety glasses, lab coats and gloves.

SDS is particularly gnarly if it's inhaled. If you're making a solution from powdered SDS, use a dust mask and/or weigh it out in a hood.
Make Amount100 µL of yeast lysis solution by mixing the following in a 1.7 ml microcentrifuge tube:
  • Amount70 µL H2O
  • Amount20 µL ReagentLithium Acetate DihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #L4158 solution, Concentration1 Molarity (M)
  • Amount10 µL ReagentSodium dodecyl sulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #436143-25G solution, Concentration10 Mass / % volume





Choose a yeast colony to analyze and circle it on the bottom of the petri dish. A large one is best.
Using a micropipette tip, scrape some of the colony off and resuspend it in the lysis solution. Vortex vigorously until the colony is mixed completely into the lysis solution.
Note
THIS IS NOT A CASE WHERE MORE IS BETTER. Your solution should be slightly cloudy. If it is quite "thick", then try again.

Incubate at Temperature70 °C for Duration00:05:00

5m
Add Amount300 µL of ReagentEthanol (100%, Molecular Biology Grade)Fisher ScientificCatalog #BP2818500 and vortex briefly. Centrifuge Centrifigation21000 x g, 00:03:00 .
Note
Make sure the centrifuge is balanced!



3m
Using a P-1000 micropipettor, carefully aspirate the supernatant and discard as biological waste. Do not disturb the pellet.
AddAmount500 µL of Concentration70 % (v/v) ReagentEthanol (100%, Molecular Biology Grade)Fisher ScientificCatalog #BP2818500 to the microcentrifuge tube. Centrifuge Centrifigation21000 x g, 00:03:00 .
Note
Make sure the centrifuge is balanced!


3m
Using a P-1000 micropipettor, carefully aspirate the supernatant and discard as biological waste. Do not disturb the pellet. Try to get as much of the ethanol off as you can.
Let the pellet dry by leaving it in a Temperature42 °C dry bath for Duration00:15:00 . Leave the cap open.
Note
If you don't have a dry bath, just leave the tube (cap open) at room temperature. Extend the time to Duration00:30:00



15m
Add Amount100 µL of ReagentTE Buffer and vortex to resuspend the pellet.

Centrifuge Centrifigation21000 x g, 00:00:30 to collect the cellular debris at the bottom. The DNA remains suspended in the supernatant.

30s
Label the tube and store at Temperature-20 °C , or proceed directly to PCR.

If you need to use this sample again:
  • Thaw the sample completely.
  • Vortex briefly to resuspend everything.
  • Centrifuge Centrifigation21000 x g, 00:00:30 to (re)collect the cellular debris at the bottom of the tube.

30s
Citations
Blount BA, Driessen MR, Ellis T. GC Preps: Fast and Easy Extraction of Stable Yeast Genomic DNA.
https://doi.org/10.1038/srep26863
Lõoke M, Kristjuhan K, Kristjuhan A. Extraction of genomic DNA from yeasts for PCR-based applications.
https://doi.org/10.2144/000113672