Editing Pichia EXpression (section)

== Transformation of Pichia by Electroporation (protocol 3).<br>  ==

Growth medium YPD (1% yeast extract, 2% bactopeptone, 2% dextrose)<br>Washing solution Ice-cold, sterile bidistilled water; ice-cold sterile 1 M sorbitol<br>Electroporation solution Ice-cold sterile 1 M sorbitol<br>Outgrowth medium Ice-cold sterile 1 M sorbitol<br>Outgrowth plates YPDS (1% yeast extract, 2% bactopeptone, 2% dextrose, 1 M sorbitol, 2&nbsp;% agar)<br>Cuvette 2 mm gap width 

<br>Making electrocompetent cells:<br>1. Streak Pichia cells from a glycerol stock (30&nbsp;% glycerol) onto YPD plates to grow single colonies.<br>2. Inoculate 10 ml YPD with one colony and grow 12-14 h with shaking at 30 °C to an O.D.600 of ~3.0. It is important to have healthy log phase cells.<br>3. Inoculate 250 ml YPD with an aliquot of the overnight culture to reach an O.D.600 of 0.005 and grow ~6 h to an O.D.600 of 1.0-1.3.<br>4. Harvest by centrifugation at 1,500 x g for 5 minutes at 4 °C.<br>5. Gently resuspend in 250 ml ice-cold ddH2O using a glass pipette, centrifuge as above.<br>6. Repeat washing step with 125 ml ice-cold ddH2O.<br>7. Resuspend in 20 ml ice-cold sorbitol and centrifuge as above. Resuspend in 0.5 ml of 1 M sorbitol and keep on ice.<br>Use the cells that day.<br>Electroporation of cells:<br>1. Add 10 μl DNA (5-10 μg) to 80 μl of electrocompetent cells. Gently mix with a blue-tip pipette several times. Transfer mixture into a prechilled cuvette and incubate on ice for 5 minutes.<br>2. Wipe moisture from the cuvette and insert the cuvette into the device.<br>3. Electroporation:<br>Mode Prokaryotes “¡“<br>Voltage (V) 1,500 V<br>Time constant (t) 5 ms<br>4. Immediately add 1 ml of ice-cold sorbitol to the cuvette. Transfer sample to sterile 15 ml tube and incubate for 2 hours without shaking at 30 °C.<br>5. Spread 200 μl aliquots on YPDS plates and incubate for 3 days at 30 °C until colonies appear. Number of colonies is increased about tenfold by adding 1 ml YPD and shaking for 3 hours before spreading thus incubating for a total of 5<br>hours. 

Expected results: 

Transformation efficiency up to 2 x 102 transformants/μg of DNA.<br> 

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