---++ Protocol for harvesting phusion protein. ---+++ Materials needed: Glycerol stock of transformed E. coli cells expressing the His-tagged protein of interest. <br> _The strain used is named EQ458. It is located in common species box; this is a Rosetta 2 (DE3) E. coli strain containing 6his-phusion-pET28 plasmid. The plasmid is KanR and the strain itself is CamR. The frozen stock is overnight growth of single colony. [[http://www.emdmillipore.com/life-science-research/rosetta-2de3-competent-cells/EMD_BIO-71397/p_brGb.s1OagkAAAEjQxl9.zLX][link to strain]]_ <br> LB medium. [[http://barricklab.org/twiki/bin/view/Lab/ProtocolsRecipesLuriaBertani][LB recipe]]<br> Chloramphenicol stock. [[http://barricklab.org/twiki/bin/view/Lab/ProtocolsAntibioticStockSolutions][Cam recipe]]<br> Kanamycin stock. [[http://barricklab.org/twiki/bin/view/Lab/ProtocolsAntibioticStockSolutions][Kan recipe]]<br> Refrigerated centrifuge. <br> FRENCH laboratory press. <br> IPTG, 100 mM stock. Dissolve 2.38 g IPTG in 100 mL deionized water. Filter sterilize and store at -20 C. <br> Ni-NTA resin spin column. ThermoFisher HiPur Ni-NTA spin columns, 1ml (cat # 88225). <br> 30k cutoff Amicon ultra-filtration membrane. Amicon Ultra-15 centrifugel filter units from Millipore (cat # UFC903008). <br> Lysis Buffer: <br> * 50 mM NaH2PO4 <br> * 300 mM NaCl <br> * 10mM Imidazole <br> * Adjust pH to 8.0 using NaOH <br> Wash Buffer: <br> * 50 mM NaH2PO4 <br> * 300 mM NaCl <br> * 20mM Imidazole <br> * Adjust pH to 8.0 using NaOH <br> Elution Buffer: <br> * 50 mM NaH2PO4 <br> * 300 mM NaCl <br> * 250 mM Imidazole <br> * Adjust pH to 8.0 using NaOH <br> Polymerase storage buffer: <br> * 50% Glycerol <br> * 100 mM Tris/HCl pH 8.0 <br> * 0.2 mM EDTA <br> * 2 mM DTT <br> * 0.2% NP-40; nonionic detergent <br> * 0.2% Tween20 <br> ---+++ Procedure: (for 2 x 500 mL culture) * Streak LB plate supplemented with Kan and Cam using frozen stock. Growth overnight at 37 C. <br> * Inoculate 1.5 mL of LB broth supplemented with Kan and Cam with single colony from streak plate. <br> * Grow overnight at 37 C shaking at 200 rpm (????). <br> * Use x uL of overnight culture to inoculate 500 mL of supplemented LB broth, grow as before for 3-4 hours until logarithmic phase is reached. Also, set up a control culture which will serve as an uninduced control. <br> * Induce the cultures to express proteins by adding IPTG at a final concentration of 1.0 mM (? is this correct?) followed by overnight growth (what is total time) at 18 C. <br> * Collect cells by centrifugation. Conditions as follows: 4 C, at 10,000 x g for 15 mins. <br> * Resuspend with 3 mL of lysis buffer and place tubes in cold room bath shaker (at dial 6 ?????) for ~ 2 hours. <br> * French press; use the full cell holding (10 mL - 35 mL), and use 1500 psi pressure. <br> * Lyse cells, heat denature at 70 C for about 15 mins. <br> * Spin down, 3700 - 4000 x g for 15 mins. <br> * Collect and keep supernatant (SN) for IMAC purifications. <br> ---++++ IMAC purification: * Prepare a 1 mL Ni-NTA resin column. <br> * Saturate column with 5x lysis buffer. <br> * Bind with 1:1 lysis buffer:SN sample. <br> * Wash with 5x wash buffer. <br> * Elute with elution buffer and collect samples after the first 1 mL flow through. <br> * Use 30k cutoff Amicon ultra-filtration membrane (???how do you use this???). <br> * Top-off with storage buffer. <br> * Spin at 4000 x g for 2.5 hours or so until volume is close to ~ 1 mL. <br>
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Topic revision: r1 - 2014-04-11 - 20:08:35 - Main.CraigBarnhart
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