Editing Pichia EXpression (section)

=== Rapid Transformation with yeast grown on plate<br> ===

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1. Prepare a transformation master mix. The following ingredients provide enough reagents for'''five''' transformation reactions. Combine in a microcentrifuge tube:

100 μL sterile 2 M lithium acetate (freshly prepared)<br>400 μL sterile 50% PEG-3350<br>4 μL 2-mercaptoethanol (STINKY!! add this in the fume hood!)

<br>Set up individual transformation reactions - for each transformation:

2. Add 15 μL of the denatured salmon sperm DNA (2 mg/mL) to labeled microcentrifuge tubes.

Note: It is important for the salmon sperm DNA to be single-stranded for this procedure to work well. '''Boil the DNA for 5 minutes to denature the DNA'''. Quick chill the DNA by placing it immediately on ice. Keep the DNA on ice until you are ready to use it.

3. Add 5 μL of miniprep plasmid DNA to the appropriately labeled microcentrifuge tube. 

4. Add 100 μL of transformation mix from step 1 to the microcentrifuge tube. Vortex for 10-15 seconds to mix the contents.

5. Using a sterile toothpick, scrape a large yeast colony from a YPD plate. Transfer the yeast to the microcentrifuge tube containing the transformation/DNA solution (Step 4) by twirling the toothpick several times. Be sure that the cells are uniformly suspended before proceeding.

<br>6. Incubate the transformation mixtures at 37 ̊C with shaking for 30-45 min. Plate the transformed cells on selective media (zeocin)