pD912

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	P. pastoris cells were transformed following a condensed standard protocol (58). The amounts of DNA used were adjusted depending on the vector size (see Results). After the transformation, screenings and rescreenings of the indicated numbers of transformants (Fig. 2 and ​and3)3) were performed as outlined previously (13, 28, 30).

Protocol tips
P. pastoris cells were transformed following a condensed standard protocol (58). The amounts of DNA used were adjusted depending on the vector size (see Results). After the transformation, screenings and rescreenings of the indicated numbers of transformants (Fig. 2 and ​and3)3) were performed as outlined previously (13, 28, 30).

Publication protocol

"P. pastoris cells were transformed following a condensed standard protocol (58). The amounts of DNA used were adjusted depending on the vector size (see Results). After the transformation, screenings and rescreenings of the indicated numbers of transformants (Fig. 2 and ​and3)3) were performed as outlined previously (13, 28, 30). Correct integration in the GUT1 locus (leading to glycerol auxotrophy) was performed as outlined previously (12). Notably, we did not stamp the cells on glycerol- or glucose-containing plates but rather inoculated them in liquid media in deep-well plates containing buffered minimal media (BM; 1.34% yeast nitrogen base, 4 × 10−5% biotin, 200 mM potassium phosphate buffer [pH 6.0], and either 1% glucose [in the form of dextrose; BMD] or 1% glycerol [BMG]) as the carbon source. The liquid media were inoculated with 1 μl from pregrown liquid cultures in DWPs (containing BMD medium) that were initially inoculated from solid transformation plates.

For eGFP fluorescence measurements, the cultures were diluted 20-fold (10 μl + 190 μl double-distilled H2O), and eGFP fluorescence was normalized to biomass (using OD600 measurements) to account for pipetting errors. eGFP fluorescence (excitation/emission wavelengths of 488/507 nm) was measured using a FLUOstar plate reader (BMG Labtech, Ortenberg, Germany) using ex/em 485-12/520 filters (gain setting, 1,300). Absorption at 600 nm was measured using a SpectraMax plus 384 plate reader (Molecular Devices, Germany). After subtracting background fluorescence/absorbance from diluted media, fluorescence was normalized to the OD600. Box plots depicting the results were generated with BoxPlotR (59)."

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