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Method: Yeast DNA Mini-Preps>

May 7, 1990

Jim Howe and Srini Ramachandra


Purpose:

Time required:

Special Reagents:

Procedure:

Day 1

  1. Inoculate 5 ml AHC or YPD with yeast colony of interest, then incubate at 30 degrees C for 1day if grown in YPD or for 2 days if grown in AHC medium.

Day 2

  1. Pellet cells by centrifuging at 2000 rpm for 10 minutes in J-6 centrifuge. Wash pellets twice in 10 ml of 40 mM EDTA/90 mM 2-Mercaptoethanol (2-ME), discarding the supernatant.
  2. Add 2 ml of SCE/2-ME/Lyticase mixture (see recipe below), and incubate at 37C for 2 hours to produce spheroplasts.
  3. Centrifuge spheroplasts at 2000 rpm for 10 minutes in J-6 centrifuge, then decant the supernatant.
  4. Add 700 l of lysis buffer (see recipe) and incubate at 68C for 15 minutes; vortex the sample intermittently during this time.
  5. Vortex to resuspend spheroplasts, then split this volume (approx. 1.2 ml) into 2 microcentrifuge tubes. Extract twice using 600 l of Phenol:Chloroform:Isoamyl Alcohol (1:1:0.02) by centrifuging at 12000 rpm for 10 minutes after addition, and recovering the upper, aqueous phase for the next round.
  6. Add 700 l of isopropanol to second aqueous phase; incubate at room temperature for 15 minutes, then centrifuge at 12000 rpm for 15 minutes. Discard the supernatent, then rinse the pellet with 70% ethanol. Air dry pellet or dry briefly in the vacuum concentrator.
  7. Suspend the DNA in 300 l of TE. Add 30 g RNase (0.05 units/mg). Incubate at 37 for>2 hours, with intermittent vortexing to dissolve the pellet.
  8. Pool the two microfuge tubes for each clone together, then add 600 l warm isopropanol. Incubate at room temperature for 15 minutes, then centrifuge at 12000 rpm for 15 minutes. Wash pellet in 70% ethanol, then air or vacuum dry.
  9. Resuspend in 300 l of TE, then add 750 l ethanol. Chill in dry ice bath for 10 minutes, then centrifuge at 12000 rpm for 15 minutes. Discard supernatent, wash in 70% ethanol, then air or vacuum dry.
  10. Resuspend in 200 l TE, then measure the DNA concentration on a spectrophotometer or by running out on a gel with standards. Expect approximately 20 g of DNA per 0.1g wet weight of yeast cells.

Solutions:

References:

Eric Green, pers. comm., from his protocol "Purification of genomic DNA from filters of yeast clones", 1989 (M V Olson lab).