(r4) ProtocolsReagentsPfuSso7d < Lab

<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

---+++ 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/][&laquo;PubMedCentral&raquo;]]
Barrick Lab > ProtocolList > ProtocolsReagentRecipes > ProtocolsReagentsPfuSso7d
Topic revision: r4 - 2014-05-14 - 20:43:03 - Main.JeffreyBarrick