Difference: ProtocolsReagentsPfuSso7d (18 vs. 19)

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Protocol for harvesting Pfu-Sso7d polymerase

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This protocol is for expressing and purifying the Pfu-Sso7d polymerase from E. coli [1]. A variant of this protein with an additional 65 amino acid changes 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 DNA products and typically you use higher annealing temperatures than when using Taq polymerase. The reported differences in fidelity between Phusion and Pfu-Sso7d compared to Taq are 84× and 32×, respectively. See the NEB website for a description of other key polymerase characteristics.
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This protocol is for expressing and purifying the Pfu-Sso7d polymerase from E. coli [1]. A variant of this protein with an additional 65 amino acid changes 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 DNA products and typically you use higher annealing temperatures than when using Taq polymerase. The reported differences in fidelity between Phusion and Pfu-Sso7d compared to Taq are 84 and 32, respectively. See the NEB website for a description of other key polymerase characteristics.
  Download Download 6his-Pfu-Sso7d-pET28 expression plasmid sequence (GenBank format)

Materials needed:

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  • Glycerol stock of EQ458 E. coli cells
    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 a single colony.
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  • Glycerol stock of EQ458 E. coli cells
    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 a single colony.
 
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  • IPTG, 100 mM stock. Dissolve 2.38 g IPTG in 100 mL deionized water. Filter sterilize and store at 20C.
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  • IPTG, 100 mM stock. Dissolve 2.38 g IPTG in 100 mL deionized water. Filter sterilize and store at –20C.
 
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  • 0.2% NP-40; nonionic detergent
  • 0.2% Tween20
  • 2 mM DTT (add immediately before using for storage)
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IMPORTANT: Add fresh DTT immediately before using a batch of storage buffer to store newly purified enzyme. Your fresh DTT should be newly dissolved from powder or from a 1 M stock that you store frozen at 20C to avoid oxidation. We have observed rapid loss of function of enzyme when enzyme is diluted in old storage buffer that had DTT added and was then stored at room temperature.
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IMPORTANT: Add fresh DTT immediately before using a batch of storage buffer to store newly purified enzyme. Your fresh DTT should be newly dissolved from powder or from a 1 M stock that you store frozen at –20C to avoid oxidation. We have observed rapid loss of function of enzyme when enzyme is diluted in old storage buffer that had DTT added and was then stored at room temperature.
 

Protein Expression

Scaled for 2 x 500 mL cultures

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Day 1: Revive and Isolate Colony
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Day –1: Revive and Isolate Colony
 
  • Streak LB plate supplemented with Kan and Cam from frozen stock of EQ458. Growth plate overnight at 37C.
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Day 2: Precondition
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Day –2: Precondition
 
  • Select single colony from O/N streak plate and inoculate 1.5 mL of LB broth supplemented with Kan and Cam. Grow overnight at 37 C shaking at 250 rpm.
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Day 1: Induce
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Day –1: Induce
 
  • Use 500 L 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.
  • 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.
Day 0: Harvest
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  • Remove cassette and place in beaker with fresh storage buffer. Allow to sit overnight.
  • If cassette has swollen, use syringe to remove some of the sample.
  • Open top of cassette and remove sample.
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  • Store your newly purified enzyme at 20C.
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  • Store your newly purified enzyme at –20C.
 

Assay purified phusion polymerase activity by PCR

  • IMPORTANT: You probably want to use commercial Phusion buffer (NEB Cat #B0518S) for your reactions. It is a proprietary formulation that gives MUCH better enzyme performance.
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  • Template for this assay is the 6his-pfu-sso7d-pET28 plasmid encoding the phusion polymerase.
  • To estimate the activity of your purified Phusion, create a dilution series of purified polymerase in water ranging from 1:200 to 1:10, and compare to NEB's stock.
  • NEB stock is viscous; for an accurate comparison to the purified Phusion, ensure you are pipetting sufficient volumes to maintain accuracy.
  • After measuring the activity of your main Phusion stock, be sure to use storage buffer that has fresh DTT added to it for making any working dilutions of your original high concentration stock that will be stored for long periods of time at 20C.
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  • Template for this assay is the 6his-pfu-sso7d-pET28 plasmid encoding the polymerase.
  • To estimate the activity, create a dilution series of purified polymerase in water ranging from 1:200 to 1:10, and compare to NEB's stock.
  • NEB stock is viscous; for an accurate comparison to the purified polymerase, ensure you are pipetting sufficient volumes to maintain accuracy.
  • After measuring the activity of your main stock, be sure to use storage buffer that has fresh DTT added to it for making any working dilutions of your original high concentration stock that will be stored for long periods of time at –20C.
  Primer sequences (position with reference to sequence in file above):
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Lane 1 2 3 4 5
Phusion dilution 25 50 100 NEB (neat) H20
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Phusion 2kb.jpg
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Phusion 2kb.jpg
 

Limitations of purified polymerase

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We have noticed reduced amplification of longer transcripts (>4kb) using our purified polymerase compared to the commercial enzyme.
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We have noticed reduced amplification of longer transcripts (>4kb) using our purified polymerase compared to the commercial enzyme.
 
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7kb_4kb.pdf
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7kb_4kb.pdf
 

References

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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: 11971207. «PubMedCentral»
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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. PubMedCentral
 
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