<noautolink> ---++ Protocol for harvesting _pfu-sso7d_ (aka Phusion) polymerase This protocol is for expressing and purifying the high fidelity _pfu-sso7d_ polymerase [1] from _E. coli_. This protein is sold as Phusion polymerase by New England Biolabs. This _pfu_ variant has the _sso7d_ processivity-enhancing domain attached that increases its speed and processivity. It generates blunt-end products and typically you use higher annealing temperatures than when using _taq_. See the [[https://www.neb.com/products/m0530-phusion-high-fidelity-dna-polymerase][NEB website]] for a description of other key enzyme characteristics. ---+++ Materials needed: _The strain used is named EQ458. It is located in common species box; this is a Rosetta 2 (DE3) E. coli strain containing 6his-pfu-sso7d-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> * Glycerol stock of transformed E. coli cells expressing the His-tagged protein of interest. <br> * LB medium. [[http://barricklab.org/twiki/bin/view/Lab/ProtocolsRecipesLuriaBertani][Link to LB recipe]]<br> * Chloramphenicol stock. [[http://barricklab.org/twiki/bin/view/Lab/ProtocolsAntibioticStockSolutions][Link to Cam recipe]]<br> * Kanamycin stock. [[http://barricklab.org/twiki/bin/view/Lab/ProtocolsAntibioticStockSolutions][Link to Kan recipe]]<br> * Refrigerated centrifuge. <br> * Spectrophotometer and cuvettes. <br> * FRENCH laboratory press. Georgiou lab, MBB, 3.310, ask before using.<br> * IPTG, 100 mM stock. Dissolve 2.38 g IPTG in 100 mL deionized water. Filter sterilize and store at -20 C. <br> * Disposable plastic columns. ThermoSci, cat #29922. [[http://www.piercenet.com/product/disposable-plastic-columns][Link to columns]]<br> * Ni-NTA agarose resin. Qiagen, cat #30210, 25 ml. [[http://www.qiagen.com/products/catalog/sample-technologies/protein-sample-technologies/purification-kits-and-resins/ni-nta-agarose][Link to resin]]<br> * Slide-A-Lyzer, 10k dialysis cassette G2. ThermoSci, cat# 87730. [[http://www.piercenet.com/product/slide-a-lyzer-g2-dialysis-cassettes-10k-mwco][Link to cassette]]<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> * 40mM 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: Make 3-4 Liters<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 cultures) * Streak LB plate supplemented with Kan and Cam using frozen stock. Growth plate overnight at 37 C. (Day -3) <br> * Select single colony from O/N streak plate and inoculate 1.5 mL of LB broth supplemented with Kan and Cam. (Day -2) <br> * Grow overnight at 37 C shaking at 250 rpm. <br> * Use 500 uL of overnight culture to inoculate 500 mL of supplemented LB broth (in 2 L flask), grow as before for ~ 3-4 hours until an OD600 of between 0.4 and 0.6 is reached. (Day -1) <br> * Induce the cultures to express proteins by adding IPTG at a final concentration of 0.5 mM (2.5 mL per 500 mL) followed by overnight growth at 18 C, 250 rpm. <br> * Collect cells by centrifugation. Conditions as follows: 4 C, at 10,000 x g for 15 mins. (Day 0) <br> * Resuspend each cell pellet with 3 mL of lysis buffer and combine tubes together, mix well using pipette. <br> * French press; use the full cell holding (10 mL - 35 mL), and use 1500 psi pressure. <br> * Collect and reintroduce into french press 1x. <br> * Heat denature at 70 C for about 15 mins. <br> * Spin down, 10,000 x g for 30 mins. <br> * Collect and keep supernatant (SN) for IMAC purifications. <br> * Syringe filter the SN. <br> ---++++ Immobilized metal ion affinity chromatography (IMAC) purification: (note: save portions at each step for protein gel). <br> * Prepare a 1 mL Ni-NTA resin column. <br> * Saturate column with 5x the column volume (so 5 mL) of lysis buffer. Repeat this step twice. <br> * Bind with 1:1 lysis buffer:SN sample. <br> * Wash with 1x column volume of lysis buffer. <br> * Wash with 5x (column volume) of wash buffer. <br> * Elute with 3 mL of elution buffer and collect all 3 mL of elution. <br> ---+++ Dialysis: [[http://www.piercenet.com/guide/overview-slide-a-lyzer-g2-dialysis-cassettes][Link to dialysis cassette use]] <br> * Place dialysis cassette into storage buffer for 2 mins. <br> * Remove top and load dialysis cassette using a pipette or syringe. <br> * Squeeze the membrane to remove excess air. <br> * Replace top and place in beaker with 500 mL or 1 L of storage buffer. This should be done in the cold room on a stir plate. <br> * Allow to sit for 2 - 4 hours. <br> * Remove cassette and place in beaker with fresh storage buffer. Allow to sit overnight. <br> * If cassette has swollen, use syringe to remove some of the sample. <br> * Open top of cassette and remove sample. <br> * Store at -20 C. <br> ---+++ Validation phusion.jpg ---+++ References 1. Wang, Y., Prosen, D. E., Mei, L., Sullivan, J. C., Finney, M., Vander Horn, P. B. (2004) A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. _Nucleic Acids Res._ *32*: 1197–1207. [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC373405/][«PubMedCentral»]]
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Topic revision: r5 - 2014-06-02 - CraigBarnhart