Lissa1:Yeast Transformations

Day 1.
 * 1) 	Inoculate the yeast strain into 5 mL of SC selection medium and incubate overnight on a rotary shaker at 200 rpm (but 230 is fine) at 30 degrees.

Day 2.
 * 1) 	Get the OD600 of your yeast. If it looks like the reading will be over an OD of 1, do a 1:10 dilution and multiply the OD600 reading by 10.
 * 2) 	Use this to figure out how many cells you have per mL. If your OD is .1, you have 10^6 cells/mL.
 * 3) 	Transfer 50 mL of 1x YPD in to a culture flask and add 2.5*10^8 cells (figure out the volume from step b) to make a final dilution of 5*10^6 cells/mL.
 * 4) 	Incubate your flask on a rotary shaker at 200 rpm at 30 degrees until the cells have divided at least twice (you’ll know because the OD will double twice). It takes about 3-5 hours.
 * 5) 	When the OD is 2*10^7 (or it’s doubled), harvest cells by centrifuging at 3000g for 5 min (this is a setting of 2.5 on the big centrifuge). You need a large centrifuge tube for this.
 * 6) 	Wash your cells in 25 mL of sterile water, and centrifuge again, same as before.
 * 7) 	Resuspend in 1 mL of sterile water.
 * 8) 	Boil a 1 mL sample of carrier DNA (salmon sperm in our case) for 5 minutes and chill on ice…. Unless this has already been done in which case the carrier is in the -20 freezer.
 * 9) 	Transfer the cell suspension to a 1.5 mL eppendorf tube and microfuge for 30 sec. Discard the supernatant.
 * 10) 	Add water to a final volume of 1 mL and vortex to resuspend. You may need to use a different volume depending on your OD.
 * 11) 	Pipette 100 uL samples in to eppendorfs (1 per transformation), microfuge at top speed for 30 seconds and discard supernatant.
 * 12) 	Make up Transformation Mixture.
 * 13) 	Each tube will get:
 * 14) 	240 uL PEG 3500 50% w/v
 * 15) 	36 uL LiAc 1.0 M
 * 16) 	50 uL boiled SS-carrier DNA
 * 17) 	34 uL plasmid DNA plus water
 * 18) 	total = 360 uL
 * 19) 	If you are doing 5 transformations, here’s what you do:
 * 20) 	A master mix of:
 * 21) 	1440 uL PEG
 * 22) 	216 uL LiAc
 * 23) 	300 uL carrier DNA
 * 24) 	Put 326 uL of the master mix in to a tube for each of your transformations. In to the tubes you can add the appropriate plasmid/water mix to get the last 34 uL.
 * 25) 	VORTEX.
 * 26) 	Incubate in a 42 degree water bath for 40 mins.
 * 27) 	Microfuge at 13 for 30 seconds and remove supernatant.
 * 28) 	Pipette 200 uL sterile water in to each tube, stir pellet with tip and vortex.
 * 29) 	Plate appropriate amount of cell suspension on to SC selection medium.
 * 30) 	Incubate at 30 degrees for 3 or 4 days.
 * 31) 	Hope that it works!