Editing Pichia EXpression (section)

=== Rapid Transformation Protocol  ===

<br>Day 1. Inoculate the yeast strain in a 2-cm 2 patch onto YPAD agar and incubate overnight at 30 °. Alternatively, the yeast strain can be inoculated into 5 ml of liquid medium (2× YPAD or SC selection medium) and incubated on a shaker at 30 ° and 200 rpm. 

<br>Day 2<br>1. Heat a tube of cartier DNA in a boiling water bath for 5 min and then chill in ice/water. 

<br>2. Scrape a 50ul portion of yeast from the YPAD plate and suspend the cells in 1 ml of sterile water in a 1.5 ml microcentrifuge tube. The suspension will contain about 5 x 108 cells. Cells grown overnight in 2× YPAD broth will reach a titer between 1 and 2 x 108/ml; the titer in SC medium will be about 5 × 107/ml. Harvest 2 ml of a YPAD culture and 5 ml of an SC<br>culture. Note: Cells in log phase growth on agar or in liquid medium will transform with high efficiency. 

<br>3. Pellet the cells at 13,000 rpm at room temperature in a microcentrifuge for 30 sec and discard the supernatant.<br>4. Add the following components of the Transformation Mix (T Mix) to the cell pellet in the order listed:<br> 

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| Component <br> 
| Volume (ul)
|-
| PEG 3350 (50%, w/v) <br> 
| 240
|-
| Lithium acetate 1.0 M 
| 36
|-
| Boiled SS-Carrier DNA (2 mg/ml) 
| 50
|-
| Plasmid DNA (0.1 to 1/ug) plus water 
| 34
|-
| Total volume <br> 
| 360
|}

&nbsp;&nbsp;&nbsp; <br>&nbsp;<br>Be sure to vortex mix the carrier DNA before pipetting it. 

5. Incubate the tube in a water bath at 42 ° for 40 to 60 min. Many laboratory strains will yield up to 1 x 105 transformants/ug plasmid after 60 min incubation. Extending the time at 42 ° to 180 min increases the yield to &gt;1 x 10<sup>6</sup>/ug with some strains. 

<br>6. Microcentrifuge at 13,000 rpm at room temperature for 30 sec and remove the T Mix with a micropipettor.<br>7. Pipette 1.0 ml of sterile water into the tube and resuspend the cells by stirring with a micropipette tip and then vortex mixing vigorously.<br>8. Pipette 10- and 100ul samples onto plates of appropriate SC selection medium, incubate at 30 ° for 3 - 4 days, and isolate transformants. The 10-/ul<br>samples should be pipetted into 100-#1 puddles of sterile water. 

<br>This protocol can be used with cultures that have been stored at room temperature or in a refrigerator. The yield will be reduced with older cultures but will generally be sufficient to isolate a number of transformants of the desired genotype.