High-Efficiency Yeast Transformation 2.0

Pre-Steps

• Make sure that the 95C and 42C heat blocks are turned on and heated to the correct temperature before the beginning.
• Remember to take the time to think about your controls before you begin (like a mock transformation and a mix transformation) For a mix transformation use an extra flip top tube and with a mock transformation follow the protocol for an extra strain of whatever the mock is and do not add DNA to it during Step 5.
• Always label every container you use! Make it so that way another person is able to tell what is inside your tubes/containers.

1. LiOAc+CARRIER DNA: For each 10 transformations add 0.3mL 1.0 M LioAc to 30uL (freshly boiled using 95C heat block for 15 minutes and then bring to room temp using ice) 10mg/ml salmon sperm DNA in a flip-top tube.
2. GOOP: For each 10 transformations freshly mix 0.4mL 0.5 M LiOAc and 1.6mL 50% PEG in a 15mL conical tube. If you need to make 50% PEG follow the following recipe: dissolve 150g PEG3550 in 165mL ddwater and filter sterilize.

For EACH Transformation

1. Pellet yeast from 3mL of an overnight culture at 3000 rpm for 2 min in a 15ml conical tube in the tabletop centrifuge located in Summer's lab (ask nicely and they will let you use it if they aren't using it).
2. Aspirate (Remove) supernatant. Re-suspend in 3mL 0.1 M LiaOAc and re-spin at 3000 rpm for 2 min in a 15 mL conical tube in the tabletop centrifuge located in Summer's lab.
3. Aspirate Supernatant. Re-Suspend in 1mL 0.1 M LiaOAc, transfer to a fliptop tube, and re-spin at 3000rpm for 2 min in the micro-centrifuge. Make sure your tubes are closed before you start the micro-centrifuge!
4. Aspirate using P1000 Pipette. Re-suspend the yeast pellet in 30uL 0.1 M LiaOAc + Carrier DNA (boiled Salmon Sperm DNA).
5. Add 5uL DNA(e.g, PCR product that had been purified using a Qiaquick column) to your yeast pellet. If you do not know where the DNA is or what they are, ask Christine or another person in the lab for help!
6. Let your strain mix sit for 15 min, at room temp.
7. Add 150uL Goop (can vortex to mix) to your transformation.
8. Let your strain mix sit for 30 min at room temp.
9. Incubate your fliptop tube(s) in the 42C heat block for 15 min.
10. Spin yeast out slowly in the micro-centrifuge once more at the following settings (2000rpm, 2 min)
11. Aspirate supernatant and re-suspend yeast in 0.5mL YPD/YEPD.
12. Leave at 30C (incubator, not shaker) for 1 hour (or 4 hr/overnight if a drug-resistant marker is present in the strain) If certain genes need a longer time to grow, you can overnight them in the incubator.
13. Gently re-spin at 2000rpm for 2 min in the micro-centrifuge, aspirate out the YPD. Then re-suspend in 120ul water.

• Organize the plates you are using (1 YPD plate and one selected for drug resistance on YPD per strain) by putting the genome name, strains, plate type, initials and date.

Make sure your tube is on top of the plates so you don't mess up.

1. Put 100uL of the selected strain onto one YPD plate and 100uL of it on one selected plate that is being tested for drug-resistance.
2. Sterilize Hockey Stick by dipping in EtOH and placing it over flame (MAKE SURE TO DO THIS BETWEEN EVERY PLATE).
3. Spread DNA strain all over the 2 selective plates by using the Hockey Stick. This is to ensure that the strain is spread throughout the plate.
4. Sterilize Hockey Stick by dipping in EtOH and placing it over flame (MAKE SURE TO DO THIS BETWEEN EVERY PLATE).
5. Parafilm the plates and stick them in the 30C incubator. Make sure that you place plates that are labeled for the same strains together to avoid confusion.