Editing Pichia EXpression (section)

== Chemical Transformation Protocol 2<br>  ==

=== '''Reagents and Solutions'''  ===

<br>The following reagents and solutions are required for all four LiAc/SS-DNA/PEG protocols.<br><u>Lithium Acetate (1.0 M)</u>. Dissolve 5.1 g of lithium acetate dihydrate (Sigma Chemical Co. Ltd., St. Louis, MO) in 50 ml of water, sterilize by autoclaving for 15 min, and store at room temperature. 

<br><u>Polyethylene Glycol 3350 (50%, w/v)</u>. Dissolve 50 g of PEG 3350 (Sigma) in 30 ml of distilled, deionized water in a 150-ml beaker on a stirring hot plate. When the solution has cooled to room temperature, make the volume up to 100-ml in a 100-ml measuring cylinder and mix thoroughly by inversion. Pour the solution in to a suitable glass bottle and autoclave for 15 min. Store, securely capped, at room<br>temperature. Evaporation of water from the solution will increase the concentration of PEG and severely reduce the yield of transformants.<br>Single-Stranded Carrier DNA (2.0 mg/ml). Dissolve 200 mg of salmon sperm DNA (Sigma) in 100 ml of TE buffer (10 mM Tris-HCl, 1 mM Na2 EDTA, pH 8.0) by stirring at 4 ° for 1-2 hr. Store 1.0-ml samples at - 2 0 °. Denature the carrier DNA in a boiling water bath for 5 min and chill in ice/water before use. Boiled samples, stored at - 2 0 °, can be reused or reboiled three times without loss of activity. 

<u>Yeast Growth Media</u>. Yeast strains are grown up on plates of YPAD agar (YpD13 supplemented with 100 mg adenine hemisulfate per liter). The yeast cells to be transformed are usually regrown in liquid 2× YPAD medium 3 (2% Bacto-yeast extract, 4% Bacto-peptone, 4% glucose and adenine hemisulfate, 100 mg/liter). 

<br><u>SC selection medium 12</u> is adjusted to pH 5.6 with 1.0 N NaOH and autoclaved. Commercial formulations of YPD agar (Bacto YPD agar) and broth (Bacto YPD broth) media are available from Becton Dickinson Microbiology Systems (Becton Dickinson, Sparks, MD; www.bd.com). These media should be supplemented with adenine hemisulfate as above. 

=== 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. 

===  ===

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

<br>

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)



=== 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. 

<br> 

'''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. 

<br> 

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.