Method: Yeast DNA Mini-Preps>

May 7, 1990

Jim Howe and Srini Ramachandra

Purpose:

To rapidly isolate genomic DNA from yeast cells. Up to 24 YAC clone samples can be processed in one day as opposed to sucrose gradient preps which require 3 days. This method is designed for <1 g wet weight of cells. Because care is not taken to prevent shearing of the DNA, the average size of the DNA fragments is probably less than 100 kb.

Time required:

2-3 days

Special Reagents:

• Lyticase (Sigma #L 8137)
• Nonidet P-40(NP-40) nonionic detergent (Sigma, Cat.#N-3516)

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 37šC 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 µl of lysis buffer (see recipe) and incubate at 68šC 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 µl 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 µl 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 µl of TE. Add 30 µg RNase (0.05 units/mg). Incubate at 37š for>2 hours, with intermittent vortexing to dissolve the pellet.
8. Pool the two microfuge tubes for each clone together, then add 600 µl 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 µl of TE, then add 750 µl 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 µl TE, then measure the DNA concentration on a spectrophotometer or by running out on a gel with standards. Expect approximately 20 µg of DNA per 0.1g wet weight of yeast cells.

Solutions:

• 40 mM EDTA, 90 mM 2-Mercaptoethanol (2-ME)

  40.0 ml	0.5M EDTA, pH8.0
  3.8 ml	2-Mercaptoethanol (12M stock)
  456.2 ml	sterile ddH2O
  --------
  500ml	Store at room temperature.

• SCE

  		Final concentration
  2M Sorbitol	50 ml	1.0 M
  1M Sodium citrate	10 ml	0.1 M
  0.25M EDTA, pH7.0	24 ml	60 mM
  sterile ddH2O	16 ml
  	------
  	100 ml

  Filter sterilize and store at room temperature.

• SCE/2-ME/Lyticase (2 ml)
  2 ml SCE

  16 µl 2-ME

  0.2 mg Lyticase

• Miniprep Lysis Buffer

  Miniprep Lysis Buffer		Final concentration
  1M Tris.HCl, pH 7.4	5 ml	50 mM
  0.5M EDTA, pH8.0	5 ml	25 mM
  5M NaCl	10 ml	500 mM
  1M MgCl2	300 µl	3 mM
  12M 2-Mercaptoethanol	25 µl	3 mM
  Nonidet P-40	100 µl	0.1 %
  10% SDS	10 ml	1 %
  	------
  bring volume to	100 ml with sterile ddH2O.

  Filter sterilize and store at room temperature.

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