Episomal Yeast Transformation:
1) Clean benchtop with 70% ethanol and start Bunsen Burner.
2) Gather following materials:
Overnight Strains To Be Used YPD Media 10 X TE 50% PEG 3550 Salmon Sperm DNA 1 M LiOAc 0.1 M LiOAc
3) Ensure that heat blocks (95C and 42C) are turned on and heated.
4) Prepare Salmon Sperm with boiling. Cover Salmon Sperm tube with tube tab (Drawer 62) and place in 95C heat block 10-15 minutes (timer). After boiled, place on ice.
5) Prepare Master Transformation Mix in a container appropriate for volume (Amounts in Microliters):
| # Reactions | 1 | 5 | 10 | 20 |
| 10X TE Buffer | 20 | 100 | 200 | 400 |
| 1M LiOAc | 20 | 100 | 200 | 400 |
| 50% PEG 3550 | 160 | 800 | 1600 | 3200 |
| DMSO | 25 | 125 | 250 | 500 |
| Salmon Sperm DNA | 5 | 25 | 50 | 100 |
| Total | 230 | 1150 | 2300 | 4600 |
6) Label flip-top tubes (appropriate number for reactions) with stickers on top (mating type, MM####, genotype, transformation plasmid). Include a “mock” transformation (wild-type yeast that does not receive plasmid) and a “mix only” tube (checks for contamination).
7) Transfer 1 milliliter (1mL) of each strain into appropriate flip-top tubes (sterile pipetting by tipping culture tubes and flaming rim).
8) Place tubes in a balanced position in micro-centrifuge. Spin at 3600 rpm for 2 minutes (check settings to make sure speed is in rpm units).
9) Remove supernatant with 1000 microliter pipet. Do not disturb yeast pellet.
10) Gently resuspend each pellet in 150 microliters 0.1 M LiOAc (NOT 1 MOLAR…THAT WILL KILL THE CELLS FOR SURE) and centrifuge again at 3600 rpm for 2 minutes.
11) Remove supernatant with pipet set to 150 microliters.
12) Resuspend gently in 20 microliters 0.1 M LiOAc.
13) Prepare an appropriate number of fresh flip-top tubes with labeled top stickers (MM####, plasmid, genotype, mating type).
14) Transfer the following to each new tube (no plasmid in “mock,” no yeast or plasmid in “mix only”):
230 microliters Transformation Master Mix
10 microliters Resuspended Yeast
5 microliters plasmid
15) Flick each flip-top tube before allowing to incubate at room temperature for 30 minutes.
16) Transfer tubes to 42C heat block to incubate for 15 minutes.
17) Balance tubes in refrigerated centrifuge and spin at maximum speed for 2 minutes (make sure temperature is set and holding at 20C). Do not include “mix only” tube.
18) Remove supernatant and resuspend cells gently in 1mL YEPD. Incubate 1 hour in 30C incubator.
19) After incubation, centrifuge tubes at 3600 rpm for 2 minutes.
20) Remove YEPD supernatant and gently resuspend in 300 microliters Baxter (distilled) Water.
21) Label an appropriate number of plates, including plates for “mock” and “mix only.”
22) Add 150 microliters of each sample to its appropriate plate.
23) Use glass hockey stick to spread sample until dry. Sterilize stick in ethanol and fire between all plates.
24) Parafilm plates and place in 30C incubator.
25) Turn off flame and clean benchtop with 70% ethanol.