Transcription of Champion pET SUMO Protein Expression System
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Champion pET SUMO Protein Expression System
User Manual Champion pET SUMO Protein Expression System For high-level Expression and enhanced solubility of recombinant proteins in E. coli and cleavage of native Protein Catalog no. K300-01 Rev. Date: 18 June 2010 Manual part no. 25-0709 MAN0000440 ii iii Table of Contents Kit Contents and Storage ..v Accessory T7-Regulated Expression ..3 Experimental Outline ..5 Methods .. 6 Cloning Considerations ..6 Producing PCR Products ..8 Cloning into pET Transforming One Shot Mach1 -T1R Competent Cells ..10 Analyzing Expressing the PCR Product ..13 Analyzing Samples ..16 Purifying the Recombinant Fusion Using SUMO Troubleshooting ..22 Performing the Control Reactions ..25 Gel Purifying PCR Addition of 3 A-Overhangs Post-Amplification ..28 Map and Features of pET SUMO.
10X PCR Buffer 100 mM Tris-HCl, pH 8.3 (at 42°C) 500 mM KCl 25 mM MgCl 2 0.01% gelatin 100 μl dNTP Mix 12.5 mM dATP 12.5 mM dCTP 12.5 mM dGTP 12.5 mM dTTP in water, pH 8.0 10 μl continued on next page
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