Editing Pichia EXpression (section)

== Transformation of Pichia by Electroporation (protocol 2).<br>  ==

<br>Based on Invitrogen protocol 

'''Materials''' 

5-100 ug pure plasmid DNA containing gene of interest, linearized by restriction digestion. 

Vector can be linearized with SacI, PmeI, and BstX.<br>Extra YPD media 

YPDS plates (with proper antibiotic, ie, 100 and 500 ug/mL Zeocin) 

500 mL YPD Media (in a 2L baffled flask)<br>1 L cold, sterile ddH2O<br>25 mL cold, sterile 1M sorbitol<br>Electroporation cuvettes (0.2 cm gap width)<br>Incubator/shaker set at NO HIGHER THAN 30 oC. If the incubator doesn’t keep consistent heat, set it at<br>28 oC. 

A Pichia strain, streaked out on a YPD plate.<br>NOTE: Because this protocol is time-consuming, It is recommended that you do multiple transformation simultaneously<br>(ie, several different plasmids, or different amount of plasmid, etc). This will save you time in the end. 

'''Protocol'''<br>1. Prepare DNA (purify linearized DNA and elute with water). 

2. Pick a single colony of your Pichia strain to grow in 10 ml YPD overnight at 30 oC in a 125 ml sterile flask.<br>3. Innoculate 500 mL YPD in a 2 L flask with 0.1-0.5 ml of the overnight culture. Grow overnight again to an O.D. of 1.2-1.5.<br>4. Centrifuge the cells at 1500 x g for 5 minutes at 4 oC. Resuspend the pellet with 500 mL of ice-cold, sterile water.<br>5. Centrifuge the cells as in step 3, and then resuspend the pellets in 250 mL of ice-cold, sterile water.<br>6. Centrifuge the cells again, then resuspend in 20 ml of ice-cold 1M sorbitol. Transfer to a 50 ml conical tube.<br>7. Centrifuge the cells again, the resuspend the pellet inn 1 mL of ice-cold 1M sorbitol for a final volume of about 1.5 mL. Keep the cells on ice and use that day—do not store the cells!<br>8. Mix 40 μL of the cells from step 6 with linearized DNA prepared as per step 1, and then transfer to an ice-cold 0.2 cm electroporation cuvette. Be sure to tap the cells down to the bottom of the cuvette<br>9. Incubate the cuvette with the cells and DNA on ice for 5 minutes.<br>10. Pulse the cells: <br><u>Pichia: 25 uF, 200 ohm, 2000 V</u><br>11. Immediately add 1 ml of ice-cold 1M sorbitol to the cuvette. Transfer the cuvette contents to a sterile 15 ml tube.<br>12. Let the tube incubate at 30 oC without shaking for 1-2 hours.<br>13. Spread 10, 25, 50, 100, and 200 μL each on separate, labelled YPDS plates containing xx adenine<br>14. Incubate plates from 3-10 days at 30 oC until colonies form.<br><br> 

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