Barrick Lab :: ProtocolsReagentsPfuSso7d

---++ 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>
This topic: Lab > WebHome > ProtocolList > ProtocolsReagentRecipes > ProtocolsReagentsPfuSso7d
Topic revision: r1 - 2014-04-11 - CraigBarnhart