Yeast Smash & Grab DNA MiniprepM.D. Rose, F. Winston, and P. Hieter (1990) Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 1. To cell pellet in Eppendorf tube, add 0.3 g (roughly 0.3 ml) of glass beads, 0.2 ml of lysis buffer and 0.2 ml of a 1:1 mix of phenol and chloroform.2.Vortex the tube at top speed for 2 min.3. Add 0.2 ml of TE (10 mM Tris, 1 mM EDTA, pH 8.0) and vortex again for a few seconds.4. Spin the tubes for 5 min (room temperature) at top speed in an Eppendorf centrifuge.5. Transfer the aqueous (upper) phase (0.38 ml) to a fresh Eppendorf tube, using a new pipette tip for each sample. Discard the tube with the glass beads.6. Add 2 volumes of 100% ethanol at room temperature. Mix thoroughly.7. Centrifuge in Eppendorf for 2-3 min at room temperature.8. Discard the supernatant (use the aspirator; take care not to dislodge the pellet).9.Rinse the pellet with 0.5 ml of cold, 70% ethanol add the ethanol slowly down the side of the tube, then centrifuge for 3-5 sec.10. Remove the supernatant. Leave the tubes open and inverted for the pellets to dry. (Or dry the pellets under vacuum.)

Glass beads Use 425-600 micron beads. Sigma G-9268 works well for us. To prepare the beads: Pour the beads (1 kg bottle) into a 2 litre beaker. Fill the beaker with concentrated HCl until the beads are fully submerged. Let stand in a fume hood for 15 min. Wash with tap-distilled water until the pH is neutral. It helps to run the water through glass tubing jammed to the bottom of the beaker so that the water flows up from the bottom of the beaker. Transfer the beads to a baking dish and bake (overnight is good). Pour the beads into a sterile bottle. Lysis buffer: 10 mM Tris, pH 8.0 1 mM EDTA 100 mM NaCl 1% SDS 2% Triton X-100

Rather Rapid Genomic PrepHoffman and Winston, Gene, 1987

1. Grow 5 ml yeast cultures to saturation.

2. Collect cells by centrifugation and resuspend in 0.5 ml of water. Transfer cells to 1.5 ml microfuge tube and collect by a 5 second spin.

3. Pour off supernatant and briefly vortex to resuspend cells in residual liquid.

4. Add 0.2 ml of Buffer A (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA

pH 8.0), 200 µl glass beads, and 0.2 ml phenol:chloroform:isoamyl alcohol (25:24:1).

5. Vortex 3 minutes (setting #7 on a foam multi-tube vortex adaptor). Add 0.2 ml TE.

6. Spin 5 minutes. Transfer aqueous to new tube. OPTIONAL: Do a chloroform extraction.

7. Add 1 ml 100% EtOH (RT; cold EtOH will create a large, dirty pellet), invert tube to mix, and spin 2 minutes.

8. Discard supernatant, and resuspend pellet in 0.4 ml TE (no need to dry pellet).

9. Add 10 µl 4M ammonium acetate, mix, and then add 1 ml 100% EtOH and mix.

10. Spin for 2 minutes and dry pellet. Resuspend in 50 µl TE and use 10 µl per digest.

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Quick Yeast DNA Prep: Isolation of Total DNA (genomic and plasmid)

Linda Hoskins, Hahn Lab 10/16/98

1. Grow a 5 ml YPD O/N culture inoculated with a single yeast colony at 30 deg. 2. Transfer culture to a small 13 x 100 glass tube. Spin down cells 2 min. in tabletop

centrifuge. Pour off supernatant. 3. Wash cells with 5 ml H2O. Spin down cells 2 min. Pour off supernatant. Cell pellet can

be stored at -20 deg. 4. Resuspend cells in 500 ul lysis buffer by vortexing.

Lysis buffer:   20 ml:

0.1M Tris pH 8.0

  2 ml 1M Tris 8.0

50 mM EDTA

  2 ml 0.5M EDTA

1% SDS   2 ml 10% SDS

14 ml H2O

5. Add acid-washed glass beads (400-500 microns) to about 2 mm below meniscus. Vortex 30 sec. Add 25 ul 5M NaCl. Vortex 30 sec. Spin down 2 min. to decrease foam.

6. Remove lysed cells with P1000 at bottom of tube and transfer to a 1.5 ml microfuge tube. 7. Add 400 ul of TE-saturated phenol. Vortex. Spin 4 min. Transfer upper phase to a clean

1.5 ml microfuge tube (~400ul).

8. Add 400 ul phenol:chloroform (4:1). Vortex. Spin 4 min. Transfer upper phase to a clean 1.5 ml microfuge tube.

9. Add 1 ml 95% EtOH and mix. Spin 6 min. Pour off EtOH. Wash with 1 ml 70% EtOH. Vortex. Spin 6 min. Pour off EtOH. Dry pellet in hood or vacuum.

10. Resuspend in 50 ul TE (vortex, 37 deg. 10 min, vortex)

Quick Preparation of Plasmid DNA from Yeast

Adapted from A. Lorincz (BRL)

Last update 6/29/99

 

1. Pick a medium size yeast colony (~3 mm dia) and transfer to 200 ul of lysis buffer. 2. Add an equal volume of glass beads (0.45 mm dia). Mix on vortex at top speed for 1 min. 3. Add 200 ul of phenol/CHCl3 (2/1). Extract one time. 4. Ethanol precipitate DNA. Wash once with 80% ETOH. 5. Dry DNA and resuspend in 100 ul TE and use to transform E. coli (try 1.0 and 0.1 ul

DNA).

Lysis Buffer (1 ml)

100 mM NaCl

100 ul 1 M NaCl

10 mM Tris 8.0

10 ul 1 M Tris 8.0

1 mM EDTA 4 ul 0.25 M EDTA

0.1% SDS 10 ul 10% SDS

  876 ul H2O

Yeast Genomic DNA Prep

Linda Hoskins/Hahn lab Aug 18, 1997(modified from Philippsen, 1991)

Grow 10ml YPD cultures o/n. Figure out cell density; inoculate 30 ml YPD and grow o/n so that cell density is approximately 2 x 108 cells/ml the next morning.

Spin down cells in 50 ml sterile conical tubes for 10 min. Centrifugation steps are performed at 4 degrees; all other steps are carried out at rm. temp. Unless otherwise specified.

Resuspend cells in 10 ml water and spin down cells.

Resuspend cells in 3 ml of 0.9 M sorbitol, 0.1 M EDTA, 50 mM DTT, pH 7.5.

Add 0.25 mg Zymolyase dissolved in 200 ul 0.9 M sorbitol and incubate with occasional shaking at 37 degrees. Conversion of spheroplasts takes 15-120 min. depending on the strain used, on the growth medium, and on the growth phase. Check for spheroplast formation by mixing 4 ul cells and 4 ul 0.1% SDS on a microscope slide. Formation is complete when 80-90% of the cells are "ghost" cells. Compare to a slide with 4 ul cells and 4 ul sorbitol solution.

Spin spheroplasts for 5 min. and carefully discard the supernatant.

Resuspend spheroplasts in 3 ml of 50 mM Tris-HCl, 50 mM EDTA, pH 8.0, by slowly and repeatedly drawing the spheroplasts into a pipette. Then mix with 0.3 ml 10% SDS and incubate at 65 degrees for 30 min.

Add 1 ml 5 M KOAc, mix, and let sit on ice for 60 min. or longer. The white precipitate that forms consists mainly of insoluble potassium dodecyl sulfate and denatured proteins.

Transfer to a 50 ml centrifuge tube and spin at 15,000 rpm for 30 min. in a Sorvall SS34 rotor. Transfer supernatant (about 4 ml) to a 12 ml disposable centrifuge tube.

Add 4 ml ice-cold absolute ethanol. On mixing, the nucleic acids (2% DNA and 98% RNA) and some residual proteins with immediately precipitate.

Spin at 10,000 rpm for 10 min. and discard the supernatant. Wash with 4 ml 70% ethanol. Spin at 10,000 rpm for 10 min.

Resuspend in 300 ul TE, pH 7.5. Pellet will take a while to dissolve. A 10 min. incubation at 42 degrees can help. May need to let sit O/N in fridge. At this point keep a 3 ul aliquot to compare to prep after Rnase treatment.

Add 15 ul 10 mg/ml Dnase-free Rnase and incubate at 37 degrees for 30 min. The stock of Rnase is dissolved in 10 mM sodium acetate, pH 7.0, and kept at –20 degrees.

Add 300 ul phenol/chloroform (1:1) and mix by inverting. Spin for 10 min. Transfer supernatant to new tubes.

Add 15 ul 3 M NaOAc and 900 ul isopropanol. Ppt. should be immediately visible, if not, put on dry ice for 5 min. Spin for 5-10 min. Wash with 80% EtOH. Air dry pellet.

Resuspend in 100-300 ul TE, pH 7.5.

Run an aliquot of purified DNA and the aliquots of DNA before RNase treatment on a 0.7% agarose gel.

The DNA should be stored at +4 degrees or –70 degrees, not at –20 degrees. Frequent freezing and thawing should be avoided.