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[Bio101] Yeast Genomic DNA Miniprep Using A FastPrep Cell Lyser

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In this protocol

Abstract

This method is a convenient way to purify high-quality genomic DNA from yeast cells. It is suitable for PCR and other assays that require genomic DNA of higher quality.

Keywords: Genomic DNA, Yeast, Small scale, FastPrep Cell Lyser, PCR

 

Materials and Reagents

  1. 5 M Ammonium acetate (pH 7.0)
  2. Chloroform
  3. Isopropanol
  4. 70% Ethanol
  5. Lysis buffer (see Recipes)

Equipment

  1. Adapted for Fastprep machine
  2. Screw-tube
  3. Glass beads
  4. Microfuge

Procedure

  1. Grow 5 ml yeast cells overnight at 30 °C.
  2. Spin,wash once with 1 ml H2O.
  3. Resuspend in 500 μl lysis buffer.
  4. Transfer to a screw-tube with acid washed glass beads.
  5. Fastprep at 6.0 speed for 2 min.
  6. Recover liquid phase with blue tip into another tube.
  7. Add 385 μl 5 M ammonium acetate pH 7.0.
  8. Incubate 5 min at 65 °C, then 5 min on ice.
  9. Add 500 μl chloroform, vortex, spin 2 min in microfuge.
  10. Take supernatant and precipitate with 1 ml isopropanol.
  11. Incubate 5 min at room temprature, then spin 5 min.
  12. Wash pellet with 70% ethanol, dry and dissolve in 50 μl H2O.
    Note: For Southern, digest 5 μl DNA; For PCR, use 0.5-1 μl DNA. For E coli transformation, use 1-5 μl DNA.

Recipes

  1. Lysis buffer
    100 mM Tris (pH 8.0)
    50 mM EDTA
    1% SDS
    For 50 ml: 5 ml 1 M Tris, 5 ml 0.5 M EDTA, 5 ml 10% SDS
How to cite: Li, X. (2011). Yeast Genomic DNA Miniprep Using A FastPrep Cell Lyser. Bio-protocol Bio101: e64. DOI: 10.21769/BioProtoc.64.
Q&A

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cecilia ojesola
Federal university of agriculture, abeokuta
can i use sodium or potassium acetate in place of ammonium acetate?.
10/10/2013 6:09:42 AM Reply
Xiyan Li
Department of Genetics, Stanford University, USA

Potassium is almost replaceable with ammonium as cations. The difference here is the pH. NH4Ac is naturally at pH7.0 without any adjustment. But you have to add acid to KAc for pH7.0, which changes the overall concentration of acetate.
Besides, ammonium acetate is volatile, thus "cleaner" than potassium for downstream applications.

Xiyan

10/10/2013 9:58:30 PM


Awesome protocol! It also works for Actinomycetes...
2/28/2013 7:25:52 AM Reply
Hats off to whoever wrote this up and posetd it.
9/3/2012 5:33:30 PM Reply