Editing Pichia EXpression (section)

=== Transformation<br>  ===

1, Mix 4 μL (50-100 ng) of linearized plasmid DNA with 40 μL of competent cells in an electroporation cuvette. Incubate for 2 minutes on ice.<br><br> 

2,&nbsp; Electroporate samples using the following parameters:<br>(i) ECM 630 electroporator (BTX, San Diego, CA USA): cuvette gap, 2.0 mm, charging voltage, 1500 V; resistance, 200Ω; capacitance, 50 μF.<br> 

<br>(ii) Gene Pulsar II electroporator (Bio-Rad Laboratories, Hercules, CA, USA); cuvette gap, 2.0 mm, charging voltage, 1500 V; resistance, 200Ω; capacitance, 25 μF.<br><br> 

3,&nbsp; Immediately after electroporation, resuspend samples in 0.5 mL 1.0 M Sorbitol and 0.5 mL YPD. Incubate in a 30°C shaker for 1 hour and then plate 100-200 μL onto YPDS agar media with increasing concentrations of Zeocin ('''200 to 1000''' μg/ml Zeocin) or 300 μg/ml Geneticin for selection of multi-copy integrants.<br> 

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use two plates: '''500''' ug/ml, '''1000 '''ug/ml<br> 

<br>4, For higher colony numbers, after the outgrowth step, centrifuge the culture tubes at 1000 x g, pour off the supernatant, and resuspend the pellet in the remaining droplet of media. Divide the suspension between the two levels of antibiotic plates and spread the culture out onto the plate.<br> 

<br>5, Incubate plates at 30°C for 2 to 3 days, until colonies are well formed. There may be some background growth visible, but definite colonies will also be present.<br><br>