Yeast Transformation

There are several methods available for transforming yeast cells. For high efficiency transformation I use the Geitz method and add DMSO. Otherwise I just grow some cells and use lithium acetate as described below.

Library Transformations Scale provides cells for 8 transformations:

inoculate 3-5 ml O/N with cells from a fresh plate and shake overnight at 30 C
measure OD600 of the O/N and inoculate a fresh 50 ml culture to OD600 0.05
- note: OD is strain specific. My cells (YPH499) OD600 1.0 = 4 x 10exp7/ml
grow cells at 30 C until OD600 0.8
pellet cells
pour off sup
resuspend in 25 ml H20
pellet cells
resuspend in 1 ml 100 mM LiOAc
take to eppendorf tube
pellet cells
resuspend to 400 ul total volume 100 mM LiOAc (add 300 ul)
aliquot 50 ul per transformation

add:	
	240 ul 50% PEG 3000
	 36 ul 1 M LiOAc
	 25 ul 2 mg/ml ssDNA
	 50 ul (1 ug or less) plasmid in water

vortex to mix contents
incubate at 30 C, 30 minutes
add 33 ul DMSO
heat shock at 45 C, 15 minutes
add 500 ul liquid media, mix
pellet cells
resuspend in H20 or media and plate

Should Yield 10exp5/ug



Regular Transformation: ( "just to get transformants" but still good efficiency )

	inoculate 5 ml O/N in glass tube
	shake approx 24 hours, 30 C
	take OD 600 (od¹s are typically 0.5 to 4.0 depending on media)
	take 1 - 1.4 ml cells to eppendorf tube
	pellet cells (spin 4k, 2 min)
	remove sup.
	add:
		240 ul 50% PEG4000
		 36 ul 1M LiOAc, pH 7.5
		 25 ul 2 mg/ml ssDNA
		approx 1 ug plasmid

	vortex, or break up cell pellet by pipetting
	incubate 30 C, 30 minutes
	add 33 ul DMSO
	heat shock 42šC, 15 min.
	add 800 ul H20
	vortex
	spin 4k, 2 min.
	resuspend in 100 ul

usually yields 10exp4/ug so plate accordingly

Chris Seidel