Lazy bones transformation

From farabaughLab

From Joan Curcio via David Garfinkel

Patch the strain(s) to be transformed onto YPD and allow to grow to a thick patch

Put 50 µg of boiled salmon sperm DNA (5 µl of a 10 mg/ml stock) into sterile Eppendorf tubes

Add 0.5 to 1 µg of plasmid DNA (you can make a stock solution of plasmid DNA + salmon sperm DNA if you are doing large numbrers of transformations)

Add a small dab of cells from the patched strains to the 5 µl of DNA and resuspend. The dab should be enough to fill the larger end of a flat toothpick and is approximately the size of a medium yeast colony)

Add 0.5 ml of freshly made PLATE buffer (see recipe below) and vortex immediately. Leave on your bench 3 hours to overnight (it may be possible to leave the tubes over the weekend)

Heat shock for 15 minutes at 42°C

Add 0.5 ml sterile water and vortex to mix the solutions

Microfuge for 1 minute at 10 K RPM

Pour off the supernatant

Resuspend in 30 to 100 µl of sterile water. If there is any residual 0.5 X PLATE buffer on the pellet, flick the droplet off the cells; keep the tube closed so the pellet isn't lost during this flicking.

Plate onto appropriate selective medium and grow at 30°C until transformants appear.

PLATE buffer

For 5 ml 3.3 ml 50% PEG-3350 0.5 ml 10 X TE 0.5 ml 1 M LiAc 0.7 ml dH2O 10 X TE: 100 mM Tris-Cl pH 7.5, 10 mM EDTA

For 50 ml 33 ml 50% PEG-3350 5 ml 10 X TE 5 ml 1 M LiAc 7 ml dH2O