Download pEcoli Expression Systems User Manual

pEcoli Expression Systems User Manual
IV.
Workflow
Choose a pET System
• Use your cloning method – pEcoli Expression
System (Cat. No. 631417)
• Seamless, easy cloning – no restriction digestion,
no dephosphorylation, no subcloning – pEcoli Linear
Expression System (Cat. No. 631418)
Prepare pET Vector (only for
pEcoli Expression System)
• Digest with restriction enzyme(s) and
dephosphorylate
• Gel-purify vector using NucleoSpin Gel and
PCR Clean-Up (Cat. No. 740609.50)
Prepare Insert DNA
• PCR the GOI using Advantage HD
Polymerase Mix (Cat. No. 639241)
• Gel-purify or spin column-purify insert using
NucleoSpin Gel and PCR Clean-Up
(Cat. No. 740609.50), depending on
fragment quality
Clone Insert into pET Vector
• Ligate or anneal insert into pET vector if using pEcoli Expression System
• Clone insert into pET vector using In-Fusion if using pEcoli Linear
Expression System
• Transform into Stellar Electrocompetent Cells (Cat. No. 636765)
• Identify positive clones; perform colony PCR using High Fidelity PCR EcoDry
Premix (Cat. No. 639280)
• Purify plasmid DNA using NucleoSpin QuickPure (Cat. No. 740615.50) or
NucleoSpin Plasmid (Cat. No. 740588.50)
• Verify reading frame by sequencing
Transform into Expression
Host
• Transform into BL21 (DE3)
competent bacteria
(EMD Biosciences, Inc.)
• Induce and Express
the Target Protein
• Analyze Expression
• Analyze Solubility
• Scale-up Culture Size
Purify Target Protein
• HisTALON Co resin for highest purity
Extract Target Protein
• TALON xTractor Buffer
• His60 Ni resin for highest binding capacity
• EP and CEP Kits are supplied
(Cat. No. 635501)
(Cat. No. 635659)
(Cat. No. 635656)
with the extraction buffer
Figure 4. Bacterial expression and purification flowchart.
Protocol No. PT5018-1
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