Pichia Expression Kit
PROTEIN 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
U.S. Headquarters:European Headquarters:
Invitrogen Corporation Invitrogen BV
3985 B Sorrento Valley Blvd.De Schelp 12, 9351 NV Leek
San Diego, CA 92121The Netherlands
Tel:(800) 955-6288Tel:+31 (0) 594 515 175
Fax:(619) 597-6201Fax:+31 (0) 594 515 312
Internet: tech_ Internet: tech_st.nl
ii
Materials
Kit Contents Box 1: Spheroplast Module. Store at room temperature.
Reagent Amount Components
SOS media20 ml 1 M Sorbitol
0.3X YPD
10 mM CaCl2
Sterile Water 2 x 125 ml Autoclaved, deionized water
SE 2 x 125 ml 1 M Sorbitol
考研英语参考书25 mM EDTA, pH 8.0
SCE 2 x 125 ml 1 M Sorbitol
10 mM Sodium citrate buffer, pH 5.8
1 mM EDTA
1 M Sorbitol
2 x 125 ml--
illuminationCaS 2 x 60 ml 1 M Sorbitol
10 mM Tris-HCl, pH 7.5;
jenna presley
10 mM CaCl2
40% PEG 25 ml40% (w/v) PEG 3350 (Reagent grade) in water
CaT25 ml20 mM Tris-HCl, pH 7.5
20 mM CaCl2
Stab Vials: Pichia and E. coli stabs. Store at +4°C.
singlehop
Strain Amount Genotype Phenotype (Pichia only)
GS115 1 stab his4Mut+
KM71 1 stab arg4 his4 aox1::ARG4Mut S, Arg+
GS115 Albumin 1 stab HIS4Mut S
GS115 β-Gal 1 stab HIS4Mut+
TOP10F´ 1 stab F´ {pro AB, lac I q, lac Z∆M15, Tn10 (Tet R)} mcr A,
∆(mrr-hsd RMS-mcr BC), φ80lac Z∆M15, ∆lac X74,
deo R, rec A1, ara D139, ∆(ara-leu)7697, gal U, gal K,
rps L(Str R), end A1, nup G λ-.
Box 2: Spheroplast Module. Store at -20°C.
Reagent Amount Components
Zymolya10 x 20 µl 3 mg/ml Zymolya in water
(100,000 units/g lytic activity)
1 M DTT10 x 1 ml 1 M dithiothreitol in water
continued on next page
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Materials, continued
Kit Contents,
continued
服装店陈列道具Box 3: Vectors and Primers. Store at -20°C
bittyReagent Description
pHIL-D2
10 µl (1 µg/µl)
Vector for intracellular expression in Pichia
pPIC3.5
10 µl (1 µg/µl)
Vector for intracellular expression in Pichia
pHIL-S1
10 µl (1 µg/µl)Vector for creted expression in Pichia.
Us the PHO1 signal quence
pPIC9
10 µl (1 µg/µl)Vector for creted expression in Pichia.
Us the α-factor signal quence
5´ AOX1 quencing primer
20 µl (0.1 µg/µl, 15.6 pmoles/µl)
charles perrault5´-GACTGGTTCCAATTGACAAGC-3´
3´ AOX1 quencing primer
20 µl (0.1 µg/µl, 15.7 pmoles/µl)
5´-GCAAATGGCATTCTGACATCC-3´
α-Factor quencing primer
20 µl (0.1 µg/µl, 15.75 pmoles/µl)
5´-TACTATTGCCAGCATTGCTGC-3´
Media The following prepackaged media is included for your convenience. Instructions for u are provided on the package.
Media Amount Yield
YP Ba Medium 2 pouches 2 liters of YP medium
YP Ba Agar Medium 2 pouches 2 liters of YP medium
Yeast Nitrogen Ba 1 pouch500 ml of 10X YNB
N OTE The α-Factor, 5´ AOX1, and 3´ AOX1 primers, and reagents to transform Pichia by spheroplasting (Pichia Spheroplast Module) are available parately from Invitrogen.
Product Reactions or Amount Catalog no.
Pichia Spheroplast Module10 spheroplast preparations
pink是啥意思(50 transformations)
K1720-01 5´ AOX1 primer 2 µg, lyophilized N710-02
3´ AOX1 primer 2 µg, lyophilized N720-02
5´ and 3´ AOX1 primers 2 µg, each, lyophilized N740-02
α-Factor primer 2 µg, each, lyophilized N730-02
continued on next page
iv
Materials, continued
Required Equip-ment and Supplies (not provided)•30°C rotary shaking incubator
•Water baths capable of 37°C, 45°C, and 100°C
•Centrifuge suitable for 50 ml conical tubes (floor or table-top)
•Baffled culture flasks with metal covers (50 ml, 250 ml, 500 ml, 1000 ml, and 3 L)•50 ml sterile, conical tubes
• 6 ml and 15 ml sterile snap-top tubes (Falcon 2059 or similar)
•UV Spectrophotometer
•Mini agaro gel apparatus and buffers雅思机经
•Polyacrylamide Gel Electrophoresis apparatus and buffers
•Media for transformation, growth, screening, and expression (e Recipes, pages 53-58)•5% SDS solution (10 ml per transformation)
•Sterile cheecloth or gauze
•Breaking Buffer (e Recipes, page 58)
•Acid-washed glass beads (available from Sigma)
•Replica-plating equipment (optional)
•Bead Breaker™ (optional)
Invitrogen provides technical support to all registered Pichia Expression Kit urs. To
receive prompt technical rvice and answers to your questions, plea provide the
Technical Reprentative with your product rial number.
For Technical Service, plea call, write, fax or E-mail:
U.S. Headquarters:European Headquarters:
Invitrogen Corporation Invitrogen BV
3985 B Sorrento Valley Blvd.De Schelp 12, 9351 NV Leek
San Diego, CA 92121The Netherlands
Tel:(800) 955-6288Tel:+31 (0) 594 515 175
学位班Fax:(619) 597-6201Fax:+31 (0) 594 515 312
Internet: tech_ Internet: tech_st.nl
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