Pichia protocols manual
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Pichia Expression KitPROTEIN EXPRESSION A Manual of Methods for Expression of Recombinant Proteins in Pichia pastoris Catalog no. K1710-01
Pichia Expression Kit
Version F
160624
25-0043
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Materials
Kit Contents
Box 1: Spheroplast Module. Store at room temperature. Reagent SOS media Amount 20 ml Components 1 M Sorbitol 0.3X YPD 10 mM CaCl2 Autoclaved, deionized water 1 M Sorbitol 25 mM EDTA, pH 8.0 1 M Sorbitol 10 mM Sodium citrate buffer, pH 5.8 1 mM EDTA -1 M Sorbitol 10 mM Tris-HCl, pH 7.5; 10 mM CaCl2 40% (w/v) PEG 3350 (Reagent grade) in water 20 mM Tris-HCl, pH 7.5 20 mM CaCl2
Sterile Water SE SCE
2 x 125 ml 2 x 125 ml 2 x 125 ml
1 M Sorbitol CaS
2 x 125 ml 2 x 60 ml
40% PEG CaT
25 ml 25 ml
Stab Vials: Pichia and E. coli stabs. Store at +4°C. Strain GS115 KM71 GS115 Albumin GS115 β-Gal TOP10F´ Amount 1 stab 1 stab 1 stab 1 stab 1 stab Genotype his4 arg4 his4 aox1::ARG4 HIS4 HIS4 Phenotype (Pichia only) Mut + Mut S, Arg + Mut S Mut +
F´ {proAB, lacI q, lacZ∆M15, Tn10 (Tet R )} mcrA, ∆(mrr-hsdRMS-mcrBC), φ80lacZ∆M15, ∆lacX74, deoR, recA1, araD139, ∆(ara-leu)7697, galU, galK, rpsL(StrR), endA1, nupG λ-.
Box 2: Spheroplast Module. Store at -20°C. Reagent Zymolyase 1 M DTT Amount 10 x 20 µl 10 x 1 ml Components 3 mg/ml Zymolyase in water (100,000 units/g lytic activity) 1 M dithiothreitol in water continued on next page
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Materials, continued
Kit Contents, continued
Box 3: Vectors and Primers. Store at -20°C Reagent pHIL-D2 10 µl (1 µg/µl) pPIC3.5 10 µl (1 µg/µl) pHIL-S1 10 µl (1 µg/µl) pPIC9 10 µl (1 µg/µl) 5´ AOX1 sequencing primer 20 µl (0.1 µg/µl, 15.6 pmoles/µl)