Editing Pichia EXpression (section)

=== High-Efficiency Transformation Protocol  ===

<br>This protocol can be used to generate sufficient transformants in a single reaction to screen multiple yeast genome equivalents for plasmids that complement a specific mutation. It can also be used to transform integrating plasmids, DNA fragments and oligonucleotides TM for yeast genome manipulation. Finally, it is used to optimize the conditions for transformation of a particular yeast strain, for example, the transformation of a plasmid library into a two-hybrid yeast strain transformed with a bait plasmid by the rapid transformation protocol. The high efficiency protocol can also be employed to transform a yeast strain simultaneously with two different plasmids, such as the two-hybrid bait and prey plasmids. 

<br>'''Day 1'''. Inoculate the yeast strain into 5 ml of liquid medium (2x YPAD or SC selection medium) and incubate overnight on a rotary shaker at 200 rpm and 30 °. Place a bottle of 2x YPAD and a 250-ml culture flask in the incubator as well. 

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'''Day 2'''<br>1. Determine the titer of the yeast culture by pipetting 10 ul of cells into 1.0 ml of water in a spectrophotometer cuvette and measuring the OD at 600 nm. For many yeast strains a suspension containing 1 x 10<sup>6</sup> cells/ml will give an OD600 of 1.0. Alternatively, titer the culture using a hemocytometer. 

<br>2. Transfer 50 ml of the prewarmed 2x YPAD to the prewarmed culture flask and add 2.5 x 10<sup>8 </sup>cells to give 5 x 10<sup>6</sup> cells/ml. 

<br>3. Incubate the flask on a rotary or reciprocating shaker at 30 ° and 200 rpm. 

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4. When the cell titer is at least 2 × 10<sup>7</sup> cells/ml, which should take about 4 hr, harvest the cells by centrifugation at 3000g at room temperature for 5 min, wash the cells in 25 ml of sterile water and resuspend in 1 ml of&nbsp; sterile water. 

<br>Boil a 1.0-ml sample of cartier DNA for 5 min and chill in an ice/water bath while harvesting the cells. Transfer the cell suspension to a 1.5-ml microcentrifuge tube, centrifuge for 30 sec, and discard the supernatant. <br> 

Add water to a final volume of 1.0 ml and vortex-mix vigorously to resuspend the cells. 

<br>Pipette 100 ul samples (ca. 10<sup>8</sup> cells) into 1.5 ml microfuge tubes, one for each transformation, centrifuge at 13,000 rpm at room temperature for 30 sec, and remove the supematant. 

<br>Make up sufficient T Mix for the planned number of transformations plus one extra. Keep the T Mix in ice/water.<br> 

{| width="494" cellspacing="1" cellpadding="1" border="1"
|-
| <br> 
| colspan="3" | Number of transformations planned
|-
| Reagents 
| 1<br> 
| 5 (6x) 
| 10 (11x)
|-
| PEG 3350 (50%, w/v) 
| 240 ul 
| &nbsp;1440ul 
| 2640ul
|-
| Lithium acetate 1.0 M 
| 36ul 
| 216 ul 
| 396ul
|-
| Boiled SS-CarrierDNA (2 mg/ml) 
| &nbsp;50ul 
| 300 u1 
| 550ul
|-
| Plasmid DNA plus water 
| 34ul 
| 204ul 
| 374ul
|-
| Total volume <br> 
| 360ul 
| 2160 ul 
| 3960 ul
|}

<br>&nbsp;&nbsp; 

Add 360ul of T Mix to each transformation tube and resuspend the cells by vortex mixing vigorously.<br>11. Incubate the tubes in a 42 ° water bath for 40 min. 

<br>12. Microcentrifuge at 13,000 rpm at room temperature for 30 sec and remove the T Mix with a micropipettor. 

<br>13. Pipette 1.0 ml of sterile water into each tube; stir the pellet with a micropipette tip and vortex vigorously. 

<br>14. Plate appropriate dilutions of the cell suspension onto SC selection medium. 

<br>For transformation with an integrating plasmid (YIp), linear construct, or oligonucleotide, plate 200ul onto each of five plates; for a YEp, YRp, or YCp library plasmid dilute 10ul of the suspension into 1.0 ml of water and plate 10- and 100ul samples onto two plates each. The 10ul samples should be pipetted directly into 100ul puddles of sterile water on the SC<br>selection medium. 

<br>15. Incubate the plates at 30 ° for 3 to 4 days and count the number of transformants.