Publication protocol
Clean up of the linearized (BglII (Cat. No. 15213-010, Invitrogen)) plasmid was performed using the Wizard SV gel PCR clean up system (Promega). Electro competent cells for Pichia transformation were prepared as per the standard protocol outlined by the company (Invitrogen). The pPIC9K/Scom-Lac linearized DNA was transformed into competent P. pastoris GS115 (his4) strain by electroporation. The various parameters of the pulse generator were set up with the following program: Charging voltage of 1500 V, capacitance of 25 mF, resistance of 200 ohm, and pulse length of 5 ms (Gene Pulser Xcell Electroporation system, Biorad, USA). A cuvette was placed in the electroporation chamber, connected to the pulse generator and was subjected to a single pulse. Five microlitres of BglII linearized pPIC9K/Scom-Lac DNA (188 ng/mL) was added to a 40 mL aliquot of competent P. pastoris and the same was transferred into a 2 mm icecold electroporation cuvette and placed on ice. The above step was repeated with a BglII linearized pPIC9K vector DNA without insert. The vector pPIC9K without insert served as the control. Immediately, 0.5 mL of ice cold 1 M sorbitol and 0.5 mL of ice cold YPD were added and the cuvette contents transferred to a sterile micro centrifuge tube and incubated in a shaking incubator for 4 h (30∞C, 100 rpm). The reaction mixture was transferred onto Minimal Dextrose (MD) agar plates (10X Dextrose, 10XYNB, 500X Biotin) deficient in histidine, incubated at 30∞C and monitored regularly for the development of colonies. Transformed clones were subjected to antibiotic selection on YPD medium containing varying concentrations of the antibiotic G418 which confers resistance to kanamycin in Pichia.
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Manufacturer protocol
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