Protocol for harvesting Pfu-Sso7d polymerase

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 6his-Pfu-Sso7d-pET28 expression plasmid sequence (GenBank format)

Materials needed

• 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 Kan^r and the strain itself is Cam^r . The frozen stock is overnight growth of a single colony.
• LB medium (recipe)
• Chloramphenicol stock (recipe)
• Kanamycin stock (recipe])
• Refrigerated centrifuge
• Spectrophotometer and cuvettes
• French press (Ex: Georgiou lab, MBB 3.310, ask before using!)
• IPTG (100 mM) stock. Dissolve 2.38 g IPTG in 100 mL deionized water. Filter sterilize and store at –20°C.
• Disposable plastic columns. ThermoSci, cat #29922
• Ni-NTA agarose resin. Qiagen, cat #30210, 25 ml
• Slide-A-Lyzer, 10k dialysis cassette G2. ThermoSci, cat# 87730

Lysis Buffer

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 10mM Imidazole
• Adjust pH to 8.0 using NaOH

Wash Buffer

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 40mM Imidazole
• Adjust pH to 8.0 using NaOH

Elution Buffer

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 250 mM Imidazole
• Adjust pH to 8.0 using NaOH

Polymerase storage buffer Make 3-4 Liters

• 50% Glycerol
• 100 mM Tris/HCl pH 8.0
• 0.2 mM EDTA
• 0.2% NP-40; nonionic detergent
• 0.2% Tween20
• 2 mM DTT (add immediately before using for storage)

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 –20°C 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 2×500 mL cultures

Day –1: Revive and Isolate Colony

• Streak LB plate supplemented with Kan and Cam from frozen stock of EQ458. Growth plate overnight at 37°C.

• 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.

• Use 500 µL 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.

• Collect cells by centrifugation. Conditions as follows: 4°C, at 10,000 x g for 15 mins.
• Resuspend each cell pellet with 3 mL of lysis buffer and combine tubes together, mix well using pipette.
• French press; use the full cell holding (10 mL - 35 mL) and 1500 psi pressure.
• Collect and reintroduce into French press 1x.
• Heat denature at 70°C for about 15 mins.
• Spin down, 10,000 x g for 30 mins.
• Syringe filter the supernatant (0.22 µm filter).
• Proceed to IMAC purifications.

Immobilized metal ion affinity chromatography (IMAC) purification

• Prepare a 1 mL Ni-NTA resin column.
• Saturate column with 5× the column volume (so 5 mL) of lysis buffer. Repeat this step twice.
• Bind with 1:1 lysis buffer:SN sample.
• Wash with 1× column volume of lysis buffer.
• Wash with 5× (column volume) of wash buffer.
• Elute with 3 mL of elution buffer and collect all 3 mL of elution.

NOTE: This purification protocol does not perfectly purify the polymerase from all other E. coli proteins. Expect your protein sample to still be heterogeneous if you run it on a gel. Don't worry, it will have sufficient purity for DNA amplification. The heating step at 70°C during harvesting helps precipitate many E. coli proteins.

Dialysis:

• Place dialysis cassette into storage buffer for 2 mins.
• Remove top and load dialysis cassette with enzyme sample using a pipette or syringe.
• Squeeze the membrane to remove excess air.
• 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.
• Allow to sit for 2 to 4 hours.
• 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.
• Store your newly purified enzyme at –20°C.

Assay purified 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.
• The 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 Phusion or a similar polymerase.
• 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 –20°C.

Primer sequences (position with reference to sequence in file above)

Reaction Mix

* PCR Conditions* (Denaturation-Annealing-Extension repeated 30x):

Sample results for 2-kb amplicon

Limitations of purified polymerase

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

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. «PubMedCentral»

Protocol for harvesting Pfu-Sso7d polymerase

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 6his-Pfu-Sso7d-pET28 expression plasmid sequence (GenBank format)

Materials needed

• 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 Kan^r and the strain itself is Cam^r . The frozen stock is overnight growth of a single colony.
• LB medium (recipe)
• Chloramphenicol stock (recipe)
• Kanamycin stock (recipe])
• Refrigerated centrifuge
• Spectrophotometer and cuvettes
• French press (Ex: Georgiou lab, MBB 3.310, ask before using!)
• IPTG (100 mM) stock. Dissolve 2.38 g IPTG in 100 mL deionized water. Filter sterilize and store at –20°C.
• Disposable plastic columns. ThermoSci, cat #29922
• Ni-NTA agarose resin. Qiagen, cat #30210, 25 ml
• Slide-A-Lyzer, 10k dialysis cassette G2. ThermoSci, cat# 87730

Lysis Buffer

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 10mM Imidazole
• Adjust pH to 8.0 using NaOH

Wash Buffer

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 40mM Imidazole
• Adjust pH to 8.0 using NaOH

Elution Buffer

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 250 mM Imidazole
• Adjust pH to 8.0 using NaOH

Polymerase storage buffer Make 3-4 Liters

• 50% Glycerol
• 100 mM Tris/HCl pH 8.0
• 0.2 mM EDTA
• 0.2% NP-40; nonionic detergent
• 0.2% Tween20
• 2 mM DTT (add immediately before using for storage)

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 –20°C 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 2×500 mL cultures

Day –1: Revive and Isolate Colony

• Streak LB plate supplemented with Kan and Cam from frozen stock of EQ458. Growth plate overnight at 37°C.

• 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.

• Use 500 µL 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.

• Collect cells by centrifugation. Conditions as follows: 4°C, at 10,000 x g for 15 mins.
• Resuspend each cell pellet with 3 mL of lysis buffer and combine tubes together, mix well using pipette.
• French press; use the full cell holding (10 mL - 35 mL) and 1500 psi pressure.
• Collect and reintroduce into French press 1x.
• Heat denature at 70°C for about 15 mins.
• Spin down, 10,000 x g for 30 mins.
• Syringe filter the supernatant (0.22 µm filter).
• Proceed to IMAC purifications.

Immobilized metal ion affinity chromatography (IMAC) purification

• Prepare a 1 mL Ni-NTA resin column.
• Saturate column with 5× the column volume (so 5 mL) of lysis buffer. Repeat this step twice.
• Bind with 1:1 lysis buffer:SN sample.
• Wash with 1× column volume of lysis buffer.
• Wash with 5× (column volume) of wash buffer.
• Elute with 3 mL of elution buffer and collect all 3 mL of elution.

NOTE: This purification protocol does not perfectly purify the polymerase from all other E. coli proteins. Expect your protein sample to still be heterogeneous if you run it on a gel. Don't worry, it will have sufficient purity for DNA amplification. The heating step at 70°C during harvesting helps precipitate many E. coli proteins.

Dialysis:

• Place dialysis cassette into storage buffer for 2 mins.
• Remove top and load dialysis cassette with enzyme sample using a pipette or syringe.
• Squeeze the membrane to remove excess air.
• 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.
• Allow to sit for 2 to 4 hours.
• 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.
• Store your newly purified enzyme at –20°C.

Assay purified 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.
• The 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 Phusion or a similar polymerase.
• 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 –20°C.

Primer sequences (position with reference to sequence in file above)

Reaction Mix

* PCR Conditions* (Denaturation-Annealing-Extension repeated 30x):

Sample results for 2-kb amplicon

Limitations of purified polymerase

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

References

Protocol for harvesting Pfu-Sso7d polymerase

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 6his-Pfu-Sso7d-pET28 expression plasmid sequence (GenBank format)

Materials needed

• 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 Kan^r and the strain itself is Cam^r . The frozen stock is overnight growth of a single colony.
• LB medium (recipe)
• Chloramphenicol stock (recipe)
• Kanamycin stock (recipe])
• Refrigerated centrifuge
• Spectrophotometer and cuvettes
• French press (Ex: Georgiou lab, MBB 3.310, ask before using!)
• IPTG (100 mM) stock. Dissolve 2.38 g IPTG in 100 mL deionized water. Filter sterilize and store at –20°C.
• Disposable plastic columns. ThermoSci, cat #29922
• Ni-NTA agarose resin. Qiagen, cat #30210, 25 ml
• Slide-A-Lyzer, 10k dialysis cassette G2. ThermoSci, cat# 87730

Lysis Buffer

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 10mM Imidazole
• Adjust pH to 8.0 using NaOH

Wash Buffer

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 40mM Imidazole
• Adjust pH to 8.0 using NaOH

Elution Buffer

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 250 mM Imidazole
• Adjust pH to 8.0 using NaOH

Polymerase storage buffer Make 3-4 Liters

• 50% Glycerol
• 100 mM Tris/HCl pH 8.0
• 0.2 mM EDTA
• 0.2% NP-40; nonionic detergent
• 0.2% Tween20
• 2 mM DTT (add immediately before using for storage)

Protein Expression

Scaled for 2×500 mL cultures

Day –1: Revive and Isolate Colony

• Streak LB plate supplemented with Kan and Cam from frozen stock of EQ458. Growth plate overnight at 37°C.

• 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.

• Use 500 µL 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.

• Collect cells by centrifugation. Conditions as follows: 4°C, at 10,000 x g for 15 mins.
• Resuspend each cell pellet with 3 mL of lysis buffer and combine tubes together, mix well using pipette.
• French press; use the full cell holding (10 mL - 35 mL) and 1500 psi pressure.

• Heat denature at 70°C for about 15 mins.
• Spin down, 10,000 x g for 30 mins.
• Syringe filter the supernatant (0.22 µm filter).
• Proceed to IMAC purifications.

Immobilized metal ion affinity chromatography (IMAC) purification

• Prepare a 1 mL Ni-NTA resin column.
• Saturate column with 5× the column volume (so 5 mL) of lysis buffer. Repeat this step twice.
• Bind with 1:1 lysis buffer:SN sample.
• Wash with 1× column volume of lysis buffer.
• Wash with 5× (column volume) of wash buffer.
• Elute with 3 mL of elution buffer and collect all 3 mL of elution.

Dialysis:

• Place dialysis cassette into storage buffer for 2 mins.
• Remove top and load dialysis cassette with enzyme sample using a pipette or syringe.
• Squeeze the membrane to remove excess air.
• 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.
• Allow to sit for 2 to 4 hours.
• 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.
• Store your newly purified enzyme at –20°C.

Assay purified 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.
• The 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 Phusion or a similar polymerase.
• 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 –20°C.

Primer sequences (position with reference to sequence in file above)

Reaction Mix

* PCR Conditions* (Denaturation-Annealing-Extension repeated 30x):

Sample results for 2-kb amplicon

Limitations of purified polymerase

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

References

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»

Download 6his-Pfu-Sso7d-pET28 expression plasmid sequence (GenBank format)

• 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 Kan^r and the strain itself is Cam^r . The frozen stock is overnight growth of a single colony.

• Disposable plastic columns. ThermoSci, cat #29922
• Ni-NTA agarose resin. Qiagen, cat #30210, 25 ml
• Slide-A-Lyzer, 10k dialysis cassette G2. ThermoSci, cat# 87730

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 10mM Imidazole
• Adjust pH to 8.0 using NaOH

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 40mM Imidazole
• Adjust pH to 8.0 using NaOH

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 250 mM Imidazole
• Adjust pH to 8.0 using NaOH

• 50% Glycerol
• 100 mM Tris/HCl pH 8.0
• 0.2 mM EDTA
• 0.2% NP-40; nonionic detergent
• 0.2% Tween20
• 2 mM DTT (add immediately before using for storage)

• Streak LB plate supplemented with Kan and Cam from frozen stock of EQ458. Growth plate overnight at 37°C.

• 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.

• Use 500 µL 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.

• Collect cells by centrifugation. Conditions as follows: 4°C, at 10,000 x g for 15 mins.
• Resuspend each cell pellet with 3 mL of lysis buffer and combine tubes together, mix well using pipette.
• French press; use the full cell holding (10 mL - 35 mL) and 1500 psi pressure.
• Collect and reintroduce into french press 1x.
• Heat denature at 70°C for about 15 mins.
• Spin down, 10,000 x g for 30 mins.
• Syringe filter the supernatant (0.22 µm filter).
• Proceed to IMAC purifications.

• Prepare a 1 mL Ni-NTA resin column.

• Bind with 1:1 lysis buffer:SN sample.

• Elute with 3 mL of elution buffer and collect all 3 mL of elution.

• Place dialysis cassette into storage buffer for 2 mins.
• Remove top and load dialysis cassette with enzyme sample using a pipette or syringe.
• Squeeze the membrane to remove excess air.
• 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.
• Allow to sit for 2 to 4 hours.
• 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.
• Store your newly purified enzyme at –20°C.

• 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.

• 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 –20°C.

Limitations of purified polymerase

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

References

Protocol for harvesting Pfu-Sso7d polymerase

Download 6his-Pfu-Sso7d-pET28 expression plasmid sequence (GenBank format)

Materials needed:

• Refrigerated centrifuge.
• Spectrophotometer and cuvettes.
• French press. Georgiou lab, MBB, 3.310, ask before using.

• Disposable plastic columns. ThermoSci, cat #29922
• Ni-NTA agarose resin. Qiagen, cat #30210, 25 ml
• Slide-A-Lyzer, 10k dialysis cassette G2. ThermoSci, cat# 87730

Lysis Buffer:

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 10mM Imidazole
• Adjust pH to 8.0 using NaOH

Wash Buffer:

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 40mM Imidazole
• Adjust pH to 8.0 using NaOH

Elution Buffer:

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 250 mM Imidazole
• Adjust pH to 8.0 using NaOH

Polymerase storage buffer: Make 3-4 Liters

• 50% Glycerol
• 100 mM Tris/HCl pH 8.0
• 0.2 mM EDTA
• 0.2% NP-40; nonionic detergent
• 0.2% Tween20
• 2 mM DTT (add immediately before using for storage)

Protein Expression

Scaled for 2 x 500 mL cultures

• 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.

• Use 500 µL 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.

• Resuspend each cell pellet with 3 mL of lysis buffer and combine tubes together, mix well using pipette.
• French press; use the full cell holding (10 mL - 35 mL) and 1500 psi pressure.
• Collect and reintroduce into french press 1x.
• Heat denature at 70°C for about 15 mins.
• Spin down, 10,000 x g for 30 mins.
• Syringe filter the supernatant (0.22 µm filter).
• Proceed to IMAC purifications.

Immobilized metal ion affinity chromatography (IMAC) purification

• Prepare a 1 mL Ni-NTA resin column.
• Saturate column with 5x the column volume (so 5 mL) of lysis buffer. Repeat this step twice.
• Bind with 1:1 lysis buffer:SN sample.

• Wash with 5x (column volume) of wash buffer.
• Elute with 3 mL of elution buffer and collect all 3 mL of elution.

Dialysis:

• Place dialysis cassette into storage buffer for 2 mins.
• Remove top and load dialysis cassette with enzyme sample using a pipette or syringe.

• Allow to sit for 2 to 4 hours.

• Open top of cassette and remove sample.

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.

Protocol:

Conditions (Denaturation-Annealing-Extension repeated 30x):

Sample results for 2kbp amplicon:

Limitations of purified polymerase

References

Materials needed:

• LB medium. Link to LB recipe
• Chloramphenicol stock. Link to Cam recipe
• Kanamycin stock. Link to Kan recipe
• Refrigerated centrifuge.
• Spectrophotometer and cuvettes.
• French press. Georgiou lab, MBB, 3.310, ask before using.
• IPTG, 100 mM stock. Dissolve 2.38 g IPTG in 100 mL deionized water. Filter sterilize and store at –20°C.
• Disposable plastic columns. ThermoSci, cat #29922
• Ni-NTA agarose resin. Qiagen, cat #30210, 25 ml
• Slide-A-Lyzer, 10k dialysis cassette G2. ThermoSci, cat# 87730

Lysis Buffer:

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 10mM Imidazole
• Adjust pH to 8.0 using NaOH

Wash Buffer:

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 40mM Imidazole
• Adjust pH to 8.0 using NaOH

Elution Buffer:

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 250 mM Imidazole
• Adjust pH to 8.0 using NaOH

Polymerase storage buffer: Make 3-4 Liters

• 50% Glycerol
• 100 mM Tris/HCl pH 8.0
• 0.2 mM EDTA
• 0.2% NP-40; nonionic detergent
• 0.2% Tween20
• 2 mM DTT (add immediately before using for storage)

Protein Expression

Scaled for 2 x 500 mL cultures

Day –1: Revive and Isolate Colony

• Streak LB plate supplemented with Kan and Cam from frozen stock of EQ458. Growth plate overnight at 37°C.

• 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.

• Use 500 µL 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.

• Collect cells by centrifugation. Conditions as follows: 4°C, at 10,000 x g for 15 mins.
• Resuspend each cell pellet with 3 mL of lysis buffer and combine tubes together, mix well using pipette.
• French press; use the full cell holding (10 mL - 35 mL) and 1500 psi pressure.
• Collect and reintroduce into french press 1x.
• Heat denature at 70°C for about 15 mins.
• Spin down, 10,000 x g for 30 mins.
• Syringe filter the supernatant (0.22 µm filter).
• Proceed to IMAC purifications.

Immobilized metal ion affinity chromatography (IMAC) purification

• Prepare a 1 mL Ni-NTA resin column.
• Saturate column with 5x the column volume (so 5 mL) of lysis buffer. Repeat this step twice.
• Bind with 1:1 lysis buffer:SN sample.
• Wash with 1x column volume of lysis buffer.
• Wash with 5x (column volume) of wash buffer.
• Elute with 3 mL of elution buffer and collect all 3 mL of elution.

Dialysis:

• Place dialysis cassette into storage buffer for 2 mins.
• Remove top and load dialysis cassette with enzyme sample using a pipette or syringe.
• Squeeze the membrane to remove excess air.
• 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.
• Allow to sit for 2 to 4 hours.
• 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.
• Store your newly purified enzyme at –20°C.

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.
• 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 –20°C.

Primer sequences (position with reference to sequence in file above):

Protocol:

Conditions (Denaturation-Annealing-Extension repeated 30x):

Sample results for 2kbp amplicon:

Limitations of purified polymerase

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

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. «PubMedCentral»

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 DNA products and typically you use higher annealing temperatures than when using taq.

See the NEB website for a description of other key enzyme characteristics.

Expression plasmid sequence: 6his-pfu-sso7d-pET28.gbk

Materials needed:

• 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.
• LB medium. Link to LB recipe
• Chloramphenicol stock. Link to Cam recipe
• Kanamycin stock. Link to Kan recipe
• Refrigerated centrifuge.
• Spectrophotometer and cuvettes.
• French press. Georgiou lab, MBB, 3.310, ask before using.
• IPTG, 100 mM stock. Dissolve 2.38 g IPTG in 100 mL deionized water. Filter sterilize and store at –20°C.
• Disposable plastic columns. ThermoSci, cat #29922
• Ni-NTA agarose resin. Qiagen, cat #30210, 25 ml
• Slide-A-Lyzer, 10k dialysis cassette G2. ThermoSci, cat# 87730

Lysis Buffer:

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 10mM Imidazole
• Adjust pH to 8.0 using NaOH

Wash Buffer:

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 40mM Imidazole
• Adjust pH to 8.0 using NaOH

Elution Buffer:

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 250 mM Imidazole
• Adjust pH to 8.0 using NaOH

Polymerase storage buffer: Make 3-4 Liters

• 50% Glycerol
• 100 mM Tris/HCl pH 8.0
• 0.2 mM EDTA
• 0.2% NP-40; nonionic detergent
• 0.2% Tween20

Protein Expression

Scaled for 2 x 500 mL cultures

Day –1: Revive and Isolate Colony

• Streak LB plate supplemented with Kan and Cam from frozen stock of EQ458. Growth plate overnight at 37°C.

• 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.

• Use 500 µL 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.

• Collect cells by centrifugation. Conditions as follows: 4°C, at 10,000 x g for 15 mins.
• Resuspend each cell pellet with 3 mL of lysis buffer and combine tubes together, mix well using pipette.
• French press; use the full cell holding (10 mL - 35 mL) and 1500 psi pressure.
• Collect and reintroduce into french press 1x.
• Heat denature at 70°C for about 15 mins.
• Spin down, 10,000 x g for 30 mins.
• Syringe filter the supernatant (0.22 µm filter).
• Proceed to IMAC purifications.

Immobilized metal ion affinity chromatography (IMAC) purification

• Prepare a 1 mL Ni-NTA resin column.
• Saturate column with 5x the column volume (so 5 mL) of lysis buffer. Repeat this step twice.
• Bind with 1:1 lysis buffer:SN sample.
• Wash with 1x column volume of lysis buffer.
• Wash with 5x (column volume) of wash buffer.
• Elute with 3 mL of elution buffer and collect all 3 mL of elution.

Dialysis:

• Place dialysis cassette into storage buffer for 2 mins.
• Remove top and load dialysis cassette with enzyme sample using a pipette or syringe.
• Squeeze the membrane to remove excess air.
• 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.
• Allow to sit for 2 to 4 hours.
• 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.

Assay purified phusion polymerase activity by PCR

• 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.

Protocol:

Conditions (Denaturation-Annealing-Extension repeated 30x):

Sample results for 2kbp amplicon:

Limitations of purified polymerase

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. «PubMedCentral»

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 DNA products and typically you use higher annealing temperatures than when using taq.

See the NEB website for a description of other key enzyme characteristics.

Expression plasmid sequence: 6his-pfu-sso7d-pET28.gbk

Materials needed:

• 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.
• LB medium. Link to LB recipe
• Chloramphenicol stock. Link to Cam recipe
• Kanamycin stock. Link to Kan recipe
• Refrigerated centrifuge.
• Spectrophotometer and cuvettes.
• French press. Georgiou lab, MBB, 3.310, ask before using.
• IPTG, 100 mM stock. Dissolve 2.38 g IPTG in 100 mL deionized water. Filter sterilize and store at –20°C.
• Disposable plastic columns. ThermoSci, cat #29922
• Ni-NTA agarose resin. Qiagen, cat #30210, 25 ml
• Slide-A-Lyzer, 10k dialysis cassette G2. ThermoSci, cat# 87730

Lysis Buffer:

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 10mM Imidazole
• Adjust pH to 8.0 using NaOH

Wash Buffer:

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 40mM Imidazole
• Adjust pH to 8.0 using NaOH

Elution Buffer:

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 250 mM Imidazole
• Adjust pH to 8.0 using NaOH

Polymerase storage buffer: Make 3-4 Liters

• 50% Glycerol
• 100 mM Tris/HCl pH 8.0
• 0.2 mM EDTA
• 0.2% NP-40; nonionic detergent
• 0.2% Tween20
• 2 mM DTT (add immediately before use)

Protein Expression

Scaled for 2 x 500 mL cultures

Day –1: Revive and Isolate Colony

• Streak LB plate supplemented with Kan and Cam from frozen stock of EQ458. Growth plate overnight at 37°C.

• 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.

• Use 500 µL 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.

• Collect cells by centrifugation. Conditions as follows: 4°C, at 10,000 x g for 15 mins.
• Resuspend each cell pellet with 3 mL of lysis buffer and combine tubes together, mix well using pipette.
• French press; use the full cell holding (10 mL - 35 mL) and 1500 psi pressure.
• Collect and reintroduce into french press 1x.
• Heat denature at 70°C for about 15 mins.
• Spin down, 10,000 x g for 30 mins.
• Syringe filter the supernatant (0.22 µm filter).
• Proceed to IMAC purifications.

Immobilized metal ion affinity chromatography (IMAC) purification

• Prepare a 1 mL Ni-NTA resin column.
• Saturate column with 5x the column volume (so 5 mL) of lysis buffer. Repeat this step twice.
• Bind with 1:1 lysis buffer:SN sample.
• Wash with 1x column volume of lysis buffer.
• Wash with 5x (column volume) of wash buffer.
• Elute with 3 mL of elution buffer and collect all 3 mL of elution.

Dialysis:

• Place dialysis cassette into storage buffer for 2 mins.
• Remove top and load dialysis cassette with enzyme sample using a pipette or syringe.
• Squeeze the membrane to remove excess air.
• 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.
• Allow to sit for 2 to 4 hours.
• 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.
• Store at –20°C.

Assay purified phusion polymerase activity by PCR

• 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.

Primer sequences (position with reference to sequence in file above):

Protocol:

Conditions (Denaturation-Annealing-Extension repeated 30x):

Sample results for 2kbp amplicon:

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. «PubMedCentral»

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 DNA products and typically you use higher annealing temperatures than when using taq.

See the NEB website for a description of other key enzyme characteristics.

Expression plasmid sequence: 6his-pfu-sso7d-pET28.gbk

Materials needed:

• 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.
• LB medium. Link to LB recipe
• Chloramphenicol stock. Link to Cam recipe
• Kanamycin stock. Link to Kan recipe
• Refrigerated centrifuge.
• Spectrophotometer and cuvettes.
• French press. Georgiou lab, MBB, 3.310, ask before using.
• IPTG, 100 mM stock. Dissolve 2.38 g IPTG in 100 mL deionized water. Filter sterilize and store at –20°C.
• Disposable plastic columns. ThermoSci, cat #29922
• Ni-NTA agarose resin. Qiagen, cat #30210, 25 ml
• Slide-A-Lyzer, 10k dialysis cassette G2. ThermoSci, cat# 87730

Lysis Buffer:

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 10mM Imidazole
• Adjust pH to 8.0 using NaOH

Wash Buffer:

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 40mM Imidazole
• Adjust pH to 8.0 using NaOH

Elution Buffer:

• 50 mM NaH₂PO₄
• 300 mM NaCl
• 250 mM Imidazole
• Adjust pH to 8.0 using NaOH

• 50% Glycerol
• 100 mM Tris/HCl pH 8.0
• 0.2 mM EDTA

• 0.2% NP-40; nonionic detergent
• 0.2% Tween20

Protein Expression

Scaled for 2 x 500 mL cultures

Day –1: Revive and Isolate Colony

• Streak LB plate supplemented with Kan and Cam from frozen stock of EQ458. Growth plate overnight at 37°C.

• 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.

• Use 500 µL 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.

• Collect cells by centrifugation. Conditions as follows: 4°C, at 10,000 x g for 15 mins.
• Resuspend each cell pellet with 3 mL of lysis buffer and combine tubes together, mix well using pipette.
• French press; use the full cell holding (10 mL - 35 mL) and 1500 psi pressure.
• Collect and reintroduce into french press 1x.
• Heat denature at 70°C for about 15 mins.
• Spin down, 10,000 x g for 30 mins.
• Syringe filter the supernatant (0.22 µm filter).
• Proceed to IMAC purifications.

Immobilized metal ion affinity chromatography (IMAC) purification

• Prepare a 1 mL Ni-NTA resin column.
• Saturate column with 5x the column volume (so 5 mL) of lysis buffer. Repeat this step twice.
• Bind with 1:1 lysis buffer:SN sample.
• Wash with 1x column volume of lysis buffer.
• Wash with 5x (column volume) of wash buffer.
• Elute with 3 mL of elution buffer and collect all 3 mL of elution.

Dialysis:

• Place dialysis cassette into storage buffer for 2 mins.
• Remove top and load dialysis cassette with enzyme sample using a pipette or syringe.
• Squeeze the membrane to remove excess air.
• 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.
• Allow to sit for 2 to 4 hours.
• 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.
• Store at –20°C.

Assay purified phusion polymerase activity by PCR

• 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.

Primer sequences (position with reference to sequence in file above):

Protocol:

Conditions (Denaturation-Annealing-Extension repeated 30x):

Sample results for 2kbp amplicon:

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. «PubMedCentral»

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 DNA products and typically you use higher annealing temperatures than when using taq.

See the NEB website for a description of other key enzyme characteristics.

Expression plasmid sequence: 6his-pfu-sso7d-pET28.gbk

Materials needed:

• 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.
• LB medium. Link to LB recipe
• Chloramphenicol stock. Link to Cam recipe
• Kanamycin stock. Link to Kan recipe
• Refrigerated centrifuge.
• Spectrophotometer and cuvettes.
• French press. Georgiou lab, MBB, 3.310, ask before using.
• IPTG, 100 mM stock. Dissolve 2.38 g IPTG in 100 mL deionized water. Filter sterilize and store at –20°C.
• Disposable plastic columns. ThermoSci, cat #29922
• Ni-NTA agarose resin. Qiagen, cat #30210, 25 ml
• Slide-A-Lyzer, 10k dialysis cassette G2. ThermoSci, cat# 87730

Lysis Buffer:

• 300 mM NaCl
• 10mM Imidazole
• Adjust pH to 8.0 using NaOH

Wash Buffer:

• 300 mM NaCl
• 40mM Imidazole
• Adjust pH to 8.0 using NaOH

Elution Buffer:

• 300 mM NaCl
• 250 mM Imidazole
• Adjust pH to 8.0 using NaOH

• 50% Glycerol
• 100 mM Tris/HCl pH 8.0
• 0.2 mM EDTA
• 2 mM DTT
• 0.2% NP-40; nonionic detergent
• 0.2% Tween20

Protein Expression

Scaled for 2 x 500 mL cultures

Day –1: Revive and Isolate Colony

• Streak LB plate supplemented with Kan and Cam from frozen stock of EQ458. Growth plate overnight at 37°C.

• 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.

• Use 500 µL 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.

• Collect cells by centrifugation. Conditions as follows: 4°C, at 10,000 x g for 15 mins.
• Resuspend each cell pellet with 3 mL of lysis buffer and combine tubes together, mix well using pipette.
• French press; use the full cell holding (10 mL - 35 mL) and 1500 psi pressure.
• Collect and reintroduce into french press 1x.
• Heat denature at 70°C for about 15 mins.
• Spin down, 10,000 x g for 30 mins.
• Syringe filter the supernatant (0.22 µm filter).
• Proceed to IMAC purifications.

Immobilized metal ion affinity chromatography (IMAC) purification

• Prepare a 1 mL Ni-NTA resin column.
• Saturate column with 5x the column volume (so 5 mL) of lysis buffer. Repeat this step twice.
• Bind with 1:1 lysis buffer:SN sample.
• Wash with 1x column volume of lysis buffer.
• Wash with 5x (column volume) of wash buffer.
• Elute with 3 mL of elution buffer and collect all 3 mL of elution.

Dialysis:

• Place dialysis cassette into storage buffer for 2 mins.
• Remove top and load dialysis cassette with enzyme sample using a pipette or syringe.
• Squeeze the membrane to remove excess air.
• 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.
• Allow to sit for 2 to 4 hours.
• 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.
• Store at –20°C.

Assay purified phusion polymerase activity by PCR

• 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.

Primer sequences (position with reference to sequence in file above):

Protocol:

Conditions (Denaturation-Annealing-Extension repeated 30x):

Sample results for 2kbp amplicon:

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. «PubMedCentral»

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 DNA products and typically you use higher annealing temperatures than when using taq.

See the NEB website for a description of other key enzyme characteristics.

Expression plasmid sequence: 6his-pfu-sso7d-pET28.gbk

Materials needed:

• 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.
• LB medium. Link to LB recipe
• Chloramphenicol stock. Link to Cam recipe
• Kanamycin stock. Link to Kan recipe
• Refrigerated centrifuge.
• Spectrophotometer and cuvettes.
• French press. Georgiou lab, MBB, 3.310, ask before using.
• IPTG, 100 mM stock. Dissolve 2.38 g IPTG in 100 mL deionized water. Filter sterilize and store at –20°C.
• Disposable plastic columns. ThermoSci, cat #29922
• Ni-NTA agarose resin. Qiagen, cat #30210, 25 ml
• Slide-A-Lyzer, 10k dialysis cassette G2. ThermoSci, cat# 87730

Lysis Buffer:

• 50 mM NaH2PO4
• 300 mM NaCl
• 10mM Imidazole
• Adjust pH to 8.0 using NaOH

Wash Buffer:

• 50 mM NaH2PO4
• 300 mM NaCl
• 40mM Imidazole
• Adjust pH to 8.0 using NaOH

Elution Buffer:

• 50 mM NaH2PO4
• 300 mM NaCl
• 250 mM Imidazole
• Adjust pH to 8.0 using NaOH

Polymerase storage buffer: Make 3-4 Liters

• 50% Glycerol
• 100 mM Tris/HCl pH 8.0
• 0.2 mM EDTA
• 2 mM DTT
• 0.2% NP-40; nonionic detergent
• 0.2% Tween20

Protein Expression

Scaled for 2 x 500 mL cultures

Day –1: Revive and Isolate Colony

• Streak LB plate supplemented with Kan and Cam from frozen stock of EQ458. Growth plate overnight at 37°C.

• 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.

• Use 500 µL 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.

• Collect cells by centrifugation. Conditions as follows: 4°C, at 10,000 x g for 15 mins.
• Resuspend each cell pellet with 3 mL of lysis buffer and combine tubes together, mix well using pipette.
• French press; use the full cell holding (10 mL - 35 mL) and 1500 psi pressure.
• Collect and reintroduce into french press 1x.
• Heat denature at 70°C for about 15 mins.
• Spin down, 10,000 x g for 30 mins.
• Syringe filter the supernatant (0.22 µm filter).
• Proceed to IMAC purifications.

Immobilized metal ion affinity chromatography (IMAC) purification

• Prepare a 1 mL Ni-NTA resin column.
• Saturate column with 5x the column volume (so 5 mL) of lysis buffer. Repeat this step twice.
• Bind with 1:1 lysis buffer:SN sample.
• Wash with 1x column volume of lysis buffer.
• Wash with 5x (column volume) of wash buffer.
• Elute with 3 mL of elution buffer and collect all 3 mL of elution.

Dialysis:

• Place dialysis cassette into storage buffer for 2 mins.
• Remove top and load dialysis cassette with enzyme sample using a pipette or syringe.
• Squeeze the membrane to remove excess air.
• 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.
• Allow to sit for 2 to 4 hours.
• 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.
• Store at –20°C.

Assay purified phusion polymerase activity by PCR

• 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.

Primer sequences (position with reference to sequence in file above):

Protocol:

Conditions (Denaturation-Annealing-Extension repeated 30x):

Sample results for 2kbp amplicon:

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. «PubMedCentral»

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 DNA products and typically you use higher annealing temperatures than when using taq.

See the NEB website for a description of other key enzyme characteristics.

Expression plasmid sequence: 6his-pfu-sso7d-pET28.gbk

Materials needed:

• 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.
• LB medium. Link to LB recipe
• Chloramphenicol stock. Link to Cam recipe
• Kanamycin stock. Link to Kan recipe
• Refrigerated centrifuge.
• Spectrophotometer and cuvettes.
• French press. Georgiou lab, MBB, 3.310, ask before using.
• IPTG, 100 mM stock. Dissolve 2.38 g IPTG in 100 mL deionized water. Filter sterilize and store at –20°C.
• Disposable plastic columns. ThermoSci, cat #29922
• Ni-NTA agarose resin. Qiagen, cat #30210, 25 ml
• Slide-A-Lyzer, 10k dialysis cassette G2. ThermoSci, cat# 87730

Lysis Buffer:

• 50 mM NaH2PO4
• 300 mM NaCl
• 10mM Imidazole
• Adjust pH to 8.0 using NaOH

Wash Buffer:

• 50 mM NaH2PO4
• 300 mM NaCl
• 40mM Imidazole
• Adjust pH to 8.0 using NaOH

Elution Buffer:

• 50 mM NaH2PO4
• 300 mM NaCl
• 250 mM Imidazole
• Adjust pH to 8.0 using NaOH

Polymerase storage buffer: Make 3-4 Liters

• 50% Glycerol
• 100 mM Tris/HCl pH 8.0
• 0.2 mM EDTA
• 2 mM DTT
• 0.2% NP-40; nonionic detergent
• 0.2% Tween20

Protein Expression

Scaled for 2 x 500 mL cultures

Day –1: Revive and Isolate Colony

• Streak LB plate supplemented with Kan and Cam from frozen stock of EQ458. Growth plate overnight at 37°C.

• 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.

• Use 500 µL 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.

• Collect cells by centrifugation. Conditions as follows: 4°C, at 10,000 x g for 15 mins.
• Resuspend each cell pellet with 3 mL of lysis buffer and combine tubes together, mix well using pipette.
• French press; use the full cell holding (10 mL - 35 mL) and 1500 psi pressure.
• Collect and reintroduce into french press 1x.
• Heat denature at 70°C for about 15 mins.
• Spin down, 10,000 x g for 30 mins.
• Syringe filter the supernatant (0.22 µm filter).
• Proceed to IMAC purifications.

Immobilized metal ion affinity chromatography (IMAC) purification

• Prepare a 1 mL Ni-NTA resin column.
• Saturate column with 5x the column volume (so 5 mL) of lysis buffer. Repeat this step twice.
• Bind with 1:1 lysis buffer:SN sample.
• Wash with 1x column volume of lysis buffer.
• Wash with 5x (column volume) of wash buffer.
• Elute with 3 mL of elution buffer and collect all 3 mL of elution.

Dialysis:

• Place dialysis cassette into storage buffer for 2 mins.
• Remove top and load dialysis cassette with enzyme sample using a pipette or syringe.
• Squeeze the membrane to remove excess air.
• 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.
• Allow to sit for 2 to 4 hours.
• 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.
• Store at –20°C.

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. «PubMedCentral»

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 DNA products and typically you use higher annealing temperatures than when using taq.

See the NEB website for a description of other key enzyme characteristics.

Expression plasmid sequence: 6his-pfu-sso7d-pET28.gbk

Materials needed:

• 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.
• LB medium. Link to LB recipe
• Chloramphenicol stock. Link to Cam recipe
• Kanamycin stock. Link to Kan recipe
• Refrigerated centrifuge.
• Spectrophotometer and cuvettes.
• French press. Georgiou lab, MBB, 3.310, ask before using.
• IPTG, 100 mM stock. Dissolve 2.38 g IPTG in 100 mL deionized water. Filter sterilize and store at –20°C.
• Disposable plastic columns. ThermoSci, cat #29922
• Ni-NTA agarose resin. Qiagen, cat #30210, 25 ml
• Slide-A-Lyzer, 10k dialysis cassette G2. ThermoSci, cat# 87730

Lysis Buffer:

• 50 mM NaH2PO4
• 300 mM NaCl
• 10mM Imidazole
• Adjust pH to 8.0 using NaOH

Wash Buffer:

• 50 mM NaH2PO4
• 300 mM NaCl
• 40mM Imidazole
• Adjust pH to 8.0 using NaOH

Elution Buffer:

• 50 mM NaH2PO4
• 300 mM NaCl
• 250 mM Imidazole
• Adjust pH to 8.0 using NaOH

Polymerase storage buffer: Make 3-4 Liters

• 50% Glycerol
• 100 mM Tris/HCl pH 8.0
• 0.2 mM EDTA
• 2 mM DTT
• 0.2% NP-40; nonionic detergent
• 0.2% Tween20

Protein Expression

Scaled for 2 x 500 mL cultures

Day –1: Revive and Isolate Colony

• Streak LB plate supplemented with Kan and Cam from frozen stock of EQ458. Growth plate overnight at 37°C.

• 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.

• Use 500 µL 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.

• Collect cells by centrifugation. Conditions as follows: 4°C, at 10,000 x g for 15 mins.
• Resuspend each cell pellet with 3 mL of lysis buffer and combine tubes together, mix well using pipette.
• French press; use the full cell holding (10 mL - 35 mL) and 1500 psi pressure.
• Collect and reintroduce into french press 1x.
• Heat denature at 70°C for about 15 mins.
• Spin down, 10,000 x g for 30 mins.
• Syringe filter the supernatant (0.22 µm filter).
• Proceed to IMAC purifications.

Immobilized metal ion affinity chromatography (IMAC) purification

• Prepare a 1 mL Ni-NTA resin column.
• Saturate column with 5x the column volume (so 5 mL) of lysis buffer. Repeat this step twice.
• Bind with 1:1 lysis buffer:SN sample.
• Wash with 1x column volume of lysis buffer.
• Wash with 5x (column volume) of wash buffer.
• Elute with 3 mL of elution buffer and collect all 3 mL of elution.

Dialysis:

• Place dialysis cassette into storage buffer for 2 mins.
• Remove top and load dialysis cassette with enzyme sample using a pipette or syringe.
• Squeeze the membrane to remove excess air.
• 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.
• Allow to sit for 2 to 4 hours.
• 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.
• Store at –20°C.

PCR

• Perform PCRs with different dilutions of your Phusion enzyme (in storage buffer) to estimate its specific activity (units/µl).

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. «PubMedCentral»

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 DNA products and typically you use higher annealing temperatures than when using taq.

See the NEB website for a description of other key enzyme characteristics.

Expression plasmid sequence: 6his-pfu-sso7d-pET28.gbk

Materials needed:

• 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.
• LB medium. Link to LB recipe
• Chloramphenicol stock. Link to Cam recipe
• Kanamycin stock. Link to Kan recipe
• Refrigerated centrifuge.
• Spectrophotometer and cuvettes.
• French press. Georgiou lab, MBB, 3.310, ask before using.
• IPTG, 100 mM stock. Dissolve 2.38 g IPTG in 100 mL deionized water. Filter sterilize and store at –20°C.
• Disposable plastic columns. ThermoSci, cat #29922
• Ni-NTA agarose resin. Qiagen, cat #30210, 25 ml
• Slide-A-Lyzer, 10k dialysis cassette G2. ThermoSci, cat# 87730

Lysis Buffer:

• 50 mM NaH2PO4
• 300 mM NaCl
• 10mM Imidazole
• Adjust pH to 8.0 using NaOH

Wash Buffer:

• 50 mM NaH2PO4
• 300 mM NaCl
• 40mM Imidazole
• Adjust pH to 8.0 using NaOH

Elution Buffer:

• 50 mM NaH2PO4
• 300 mM NaCl
• 250 mM Imidazole
• Adjust pH to 8.0 using NaOH

Polymerase storage buffer: Make 3-4 Liters

• 50% Glycerol
• 100 mM Tris/HCl pH 8.0
• 0.2 mM EDTA
• 2 mM DTT
• 0.2% NP-40; nonionic detergent
• 0.2% Tween20

Protein Expression

Scaled for 2 x 500 mL cultures

Day –1: Revive and Isolate Colony

• Streak LB plate supplemented with Kan and Cam from frozen stock of EQ458. Growth plate overnight at 37°C.

• 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.

• Use 500 µL 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.

• Collect cells by centrifugation. Conditions as follows: 4°C, at 10,000 x g for 15 mins.
• Resuspend each cell pellet with 3 mL of lysis buffer and combine tubes together, mix well using pipette.
• French press; use the full cell holding (10 mL - 35 mL) and 1500 psi pressure.
• Collect and reintroduce into french press 1x.
• Heat denature at 70°C for about 15 mins.
• Spin down, 10,000 x g for 30 mins.
• Syringe filter the supernatant (0.22 µm filter).
• Proceed to IMAC purifications.

Immobilized metal ion affinity chromatography (IMAC) purification

• Prepare a 1 mL Ni-NTA resin column.
• Saturate column with 5x the column volume (so 5 mL) of lysis buffer. Repeat this step twice.
• Bind with 1:1 lysis buffer:SN sample.
• Wash with 1x column volume of lysis buffer.
• Wash with 5x (column volume) of wash buffer.
• Elute with 3 mL of elution buffer and collect all 3 mL of elution.

Dialysis:

• Place dialysis cassette into storage buffer for 2 mins.
• Remove top and load dialysis cassette with enzyme sample using a pipette or syringe.
• Squeeze the membrane to remove excess air.
• 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.
• Allow to sit for 2 to 4 hours.
• 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.
• Store at –20°C.

PCR

• Perform PCRs with different dilutions of your Phusion enzyme (in storage buffer) to estimate its specific activity (units/µl).

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. «PubMedCentral»

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 DNA products and typically you use higher annealing temperatures than when using taq.

See the NEB website for a description of other key enzyme characteristics.

Materials needed:

• 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.
• LB medium. Link to LB recipe
• Chloramphenicol stock. Link to Cam recipe
• Kanamycin stock. Link to Kan recipe
• Refrigerated centrifuge.
• Spectrophotometer and cuvettes.
• French press. Georgiou lab, MBB, 3.310, ask before using.
• IPTG, 100 mM stock. Dissolve 2.38 g IPTG in 100 mL deionized water. Filter sterilize and store at –20°C.
• Disposable plastic columns. ThermoSci, cat #29922
• Ni-NTA agarose resin. Qiagen, cat #30210, 25 ml
• Slide-A-Lyzer, 10k dialysis cassette G2. ThermoSci, cat# 87730

Lysis Buffer:

• 50 mM NaH2PO4
• 300 mM NaCl
• 10mM Imidazole
• Adjust pH to 8.0 using NaOH

Wash Buffer:

• 50 mM NaH2PO4
• 300 mM NaCl
• 40mM Imidazole
• Adjust pH to 8.0 using NaOH

Elution Buffer:

• 50 mM NaH2PO4
• 300 mM NaCl
• 250 mM Imidazole
• Adjust pH to 8.0 using NaOH

Polymerase storage buffer: Make 3-4 Liters

• 50% Glycerol
• 100 mM Tris/HCl pH 8.0
• 0.2 mM EDTA
• 2 mM DTT
• 0.2% NP-40; nonionic detergent
• 0.2% Tween20

Protein Expression

Scaled for 2 x 500 mL cultures

Day –1: Revive and Isolate Colony

• Streak LB plate supplemented with Kan and Cam from frozen stock of EQ458. Growth plate overnight at 37°C.

• 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.

• Use 500 µL 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.

• Collect cells by centrifugation. Conditions as follows: 4°C, at 10,000 x g for 15 mins.
• Resuspend each cell pellet with 3 mL of lysis buffer and combine tubes together, mix well using pipette.
• French press; use the full cell holding (10 mL - 35 mL) and 1500 psi pressure.
• Collect and reintroduce into french press 1x.
• Heat denature at 70°C for about 15 mins.
• Spin down, 10,000 x g for 30 mins.
• Syringe filter the supernatant (0.22 µm filter).
• Proceed to IMAC purifications.

Immobilized metal ion affinity chromatography (IMAC) purification

• Prepare a 1 mL Ni-NTA resin column.
• Saturate column with 5x the column volume (so 5 mL) of lysis buffer. Repeat this step twice.
• Bind with 1:1 lysis buffer:SN sample.
• Wash with 1x column volume of lysis buffer.
• Wash with 5x (column volume) of wash buffer.
• Elute with 3 mL of elution buffer and collect all 3 mL of elution.

Dialysis:

• Place dialysis cassette into storage buffer for 2 mins.
• Remove top and load dialysis cassette with enzyme sample using a pipette or syringe.
• Squeeze the membrane to remove excess air.
• 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.
• Allow to sit for 2 to 4 hours.
• 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.
• Store at –20°C.

PCR

• Perform PCRs with different dilutions of your Phusion enzyme (in storage buffer) to estimate its specific activity (units/µl).

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. «PubMedCentral»

Protocol for harvesting _pfu-sso7d (aka Phusion) polymerase

Materials needed:

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. «PubMedCentral»

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 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. link to strain

• Glycerol stock of transformed E. coli cells expressing the His-tagged protein of interest.
  
• LB medium. Link to LB recipe
  
• Chloramphenicol stock. Link to Cam recipe
  
• Kanamycin stock. Link to Kan recipe
  
• Refrigerated centrifuge.
  
• Spectrophotometer and cuvettes.
  
• FRENCH laboratory press. Georgiou lab, MBB, 3.310, ask before using.
  
• IPTG, 100 mM stock. Dissolve 2.38 g IPTG in 100 mL deionized water. Filter sterilize and store at -20 C.
  
• Disposable plastic columns. ThermoSci, cat #29922. Link to columns
  
• Ni-NTA agarose resin. Qiagen, cat #30210, 25 ml. Link to resin
  
• Slide-A-Lyzer, 10k dialysis cassette G2. ThermoSci, cat# 87730. Link to cassette
  

Lysis Buffer:

• 50 mM NaH2PO4
  
• 300 mM NaCl
  
• 10mM Imidazole
  
• Adjust pH to 8.0 using NaOH
  

Wash Buffer:

• 50 mM NaH2PO4
  
• 300 mM NaCl
  
• 40mM Imidazole
  
• Adjust pH to 8.0 using NaOH
  

Elution Buffer:

• 50 mM NaH2PO4
  
• 300 mM NaCl
  
• 250 mM Imidazole
  
• Adjust pH to 8.0 using NaOH
  

Polymerase storage buffer: Make 3-4 Liters

• 50% Glycerol
  
• 100 mM Tris/HCl pH 8.0
  
• 0.2 mM EDTA
  
• 2 mM DTT
  
• 0.2% NP-40; nonionic detergent
  
• 0.2% Tween20
  

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)
  
• Select single colony from O/N streak plate and inoculate 1.5 mL of LB broth supplemented with Kan and Cam. (Day -2)
  
• Grow overnight at 37 C shaking at 250 rpm.
  
• 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)
  
• 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.
  
• Collect cells by centrifugation. Conditions as follows: 4 C, at 10,000 x g for 15 mins. (Day 0)
  
• Resuspend each cell pellet with 3 mL of lysis buffer and combine tubes together, mix well using pipette.
  
• French press; use the full cell holding (10 mL - 35 mL), and use 1500 psi pressure.
  
• Collect and reintroduce into french press 1x.
  
• Heat denature at 70 C for about 15 mins.
  
• Spin down, 10,000 x g for 30 mins.
  
• Collect and keep supernatant (SN) for IMAC purifications.
  
• Syringe filter the SN.
  

Immobilized metal ion affinity chromatography (IMAC) purification: (note: save portions at each step for protein gel).

• Prepare a 1 mL Ni-NTA resin column.
  
• Saturate column with 5x the column volume (so 5 mL) of lysis buffer. Repeat this step twice.
  
• Bind with 1:1 lysis buffer:SN sample.
  
• Wash with 1x column volume of lysis buffer.
  
• Wash with 5x (column volume) of wash buffer.
  
• Elute with 3 mL of elution buffer and collect all 3 mL of elution.
  

Dialysis: Link to dialysis cassette use

• Place dialysis cassette into storage buffer for 2 mins.
  
• Remove top and load dialysis cassette using a pipette or syringe.
  
• Squeeze the membrane to remove excess air.
  
• 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.
  
• Allow to sit for 2 - 4 hours.
  
• 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.
  
• Store at -20 C.
  

Validation

https://barricklab.org/twiki/pub/Lab/ProtocolsReagentsPfuSso7d/phusion

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. «PubMedCentral»

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 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. link to strain

• Glycerol stock of transformed E. coli cells expressing the His-tagged protein of interest.
  
• LB medium. Link to LB recipe
  
• Chloramphenicol stock. Link to Cam recipe
  
• Kanamycin stock. Link to Kan recipe
  
• Refrigerated centrifuge.
  
• Spectrophotometer and cuvettes.
  
• FRENCH laboratory press. Georgiou lab, MBB, 3.310, ask before using.
  
• IPTG, 100 mM stock. Dissolve 2.38 g IPTG in 100 mL deionized water. Filter sterilize and store at -20 C.
  
• Disposable plastic columns. ThermoSci, cat #29922. Link to columns
  
• Ni-NTA agarose resin. Qiagen, cat #30210, 25 ml. Link to resin
  
• Slide-A-Lyzer, 10k dialysis cassette G2. ThermoSci, cat# 87730. Link to cassette
  

Lysis Buffer:

• 50 mM NaH2PO4
  
• 300 mM NaCl
  
• 10mM Imidazole
  
• Adjust pH to 8.0 using NaOH
  

Wash Buffer:

• 50 mM NaH2PO4
  
• 300 mM NaCl
  
• 40mM Imidazole
  
• Adjust pH to 8.0 using NaOH
  

Elution Buffer:

• 50 mM NaH2PO4
  
• 300 mM NaCl
  
• 250 mM Imidazole
  
• Adjust pH to 8.0 using NaOH
  

Polymerase storage buffer: Make 3-4 Liters

• 50% Glycerol
  
• 100 mM Tris/HCl pH 8.0
  
• 0.2 mM EDTA
  
• 2 mM DTT
  
• 0.2% NP-40; nonionic detergent
  
• 0.2% Tween20
  

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)
  
• Select single colony from O/N streak plate and inoculate 1.5 mL of LB broth supplemented with Kan and Cam. (Day -2)
  
• Grow overnight at 37 C shaking at 250 rpm.
  
• 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)
  
• 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.
  
• Collect cells by centrifugation. Conditions as follows: 4 C, at 10,000 x g for 15 mins. (Day 0)
  
• Resuspend each cell pellet with 3 mL of lysis buffer and combine tubes together, mix well using pipette.
  
• French press; use the full cell holding (10 mL - 35 mL), and use 1500 psi pressure.
  
• Collect and reintroduce into french press 1x.
  
• Heat denature at 70 C for about 15 mins.
  
• Spin down, 10,000 x g for 30 mins.
  
• Collect and keep supernatant (SN) for IMAC purifications.
  
• Syringe filter the SN.
  

Immobilized metal ion affinity chromatography (IMAC) purification: (note: save portions at each step for protein gel).

• Prepare a 1 mL Ni-NTA resin column.
  
• Saturate column with 5x the column volume (so 5 mL) of lysis buffer. Repeat this step twice.
  
• Bind with 1:1 lysis buffer:SN sample.
  
• Wash with 1x column volume of lysis buffer.
  
• Wash with 5x (column volume) of wash buffer.
  
• Elute with 3 mL of elution buffer and collect all 3 mL of elution.
  

Dialysis: Link to dialysis cassette use

• Place dialysis cassette into storage buffer for 2 mins.
  
• Remove top and load dialysis cassette using a pipette or syringe.
  
• Squeeze the membrane to remove excess air.
  
• 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.
  
• Allow to sit for 2 - 4 hours.
  
• 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.
  
• Store at -20 C.
  

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. «PubMedCentral»

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 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. link to strain

• Glycerol stock of transformed E. coli cells expressing the His-tagged protein of interest.
  
• LB medium. Link to LB recipe
  
• Chloramphenicol stock. Link to Cam recipe
  
• Kanamycin stock. Link to Kan recipe
  
• Refrigerated centrifuge.
  
• Spectrophotometer and cuvettes.
  
• FRENCH laboratory press. Georgiou lab, MBB, 3.310, ask before using.
  
• IPTG, 100 mM stock. Dissolve 2.38 g IPTG in 100 mL deionized water. Filter sterilize and store at -20 C.
  
• Disposable plastic columns. ThermoSci, cat #29922. Link to columns
  
• Ni-NTA agarose resin. Qiagen, cat #30210, 25 ml. Link to resin
  
• Slide-A-Lyzer, 10k dialysis cassette G2. ThermoSci, cat# 87730. Link to cassette
  

Lysis Buffer:

• 50 mM NaH2PO4
  
• 300 mM NaCl
  
• 10mM Imidazole
  
• Adjust pH to 8.0 using NaOH
  

Wash Buffer:

• 50 mM NaH2PO4
  
• 300 mM NaCl
  
• 40mM Imidazole
  
• Adjust pH to 8.0 using NaOH
  

Elution Buffer:

• 50 mM NaH2PO4
  
• 300 mM NaCl
  
• 250 mM Imidazole
  
• Adjust pH to 8.0 using NaOH
  

Polymerase storage buffer: Make 3-4 Liters

• 50% Glycerol
  
• 100 mM Tris/HCl pH 8.0
  
• 0.2 mM EDTA
  
• 2 mM DTT
  
• 0.2% NP-40; nonionic detergent
  
• 0.2% Tween20
  

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)
  
• Select single colony from O/N streak plate and inoculate 1.5 mL of LB broth supplemented with Kan and Cam. (Day -2)
  
• Grow overnight at 37 C shaking at 250 rpm.
  
• 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)
  
• 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.
  
• Collect cells by centrifugation. Conditions as follows: 4 C, at 10,000 x g for 15 mins. (Day 0)
  
• Resuspend each cell pellet with 3 mL of lysis buffer and combine tubes together, mix well using pipette.
  
• French press; use the full cell holding (10 mL - 35 mL), and use 1500 psi pressure.
  
• Collect and reintroduce into french press 1x.
  
• Heat denature at 70 C for about 15 mins.
  
• Spin down, 10,000 x g for 30 mins.
  
• Collect and keep supernatant (SN) for IMAC purifications.
  
• Syringe filter the SN.
  

Immobilized metal ion affinity chromatography (IMAC) purification: (note: save portions at each step for protein gel).

• Prepare a 1 mL Ni-NTA resin column.
  
• Saturate column with 5x the column volume (so 5 mL) of lysis buffer. Repeat this step twice.
  
• Bind with 1:1 lysis buffer:SN sample.
  
• Wash with 1x column volume of lysis buffer.
  
• Wash with 5x (column volume) of wash buffer.
  
• Elute with 3 mL of elution buffer and collect all 3 mL of elution.
  

Dialysis: Link to dialysis cassette use

• Place dialysis cassette into storage buffer for 2 mins.
  
• Remove top and load dialysis cassette using a pipette or syringe.
  
• Squeeze the membrane to remove excess air.
  
• 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.
  
• Allow to sit for 2 - 4 hours.
  
• 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.
  
• Store at -20 C.
  

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. «PubMedCentral»

• Glycerol stock of transformed E. coli cells expressing the His-tagged protein of interest.
  
• LB medium. Link to LB recipe
  
• Chloramphenicol stock. Link to Cam recipe
  
• Kanamycin stock. Link to Kan recipe
  
• Refrigerated centrifuge.
  
• Spectrophotometer and cuvettes.
  
• FRENCH laboratory press. Georgiou lab, MBB, 3.310, ask before using.
  
• IPTG, 100 mM stock. Dissolve 2.38 g IPTG in 100 mL deionized water. Filter sterilize and store at -20 C.
  
• Disposable plastic columns. ThermoSci, cat #29922. Link to columns
  
• Ni-NTA agarose resin. Qiagen, cat #30210, 25 ml. Link to resin
  
• Slide-A-Lyzer, 10k dialysis cassette G2. ThermoSci, cat# 87730. Link to cassette
  

Lysis Buffer:

• 50 mM NaH2PO4
  
• 300 mM NaCl
  
• 10mM Imidazole
  
• Adjust pH to 8.0 using NaOH
  

Wash Buffer:

• 50 mM NaH2PO4
  
• 300 mM NaCl
  
• 40mM Imidazole
  
• Adjust pH to 8.0 using NaOH
  

Elution Buffer:

• 50 mM NaH2PO4
  
• 300 mM NaCl
  
• 250 mM Imidazole
  
• Adjust pH to 8.0 using NaOH
  

Polymerase storage buffer: Make 3-4 Liters

• 50% Glycerol
  
• 100 mM Tris/HCl pH 8.0
  
• 0.2 mM EDTA
  
• 2 mM DTT
  
• 0.2% NP-40; nonionic detergent
  
• 0.2% Tween20
  

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)
  
• Select single colony from O/N streak plate and inoculate 1.5 mL of LB broth supplemented with Kan and Cam. (Day -2)
  
• Grow overnight at 37 C shaking at 250 rpm.
  
• 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)
  
• 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.
  
• Collect cells by centrifugation. Conditions as follows: 4 C, at 10,000 x g for 15 mins. (Day 0)
  
• Resuspend each cell pellet with 3 mL of lysis buffer and combine tubes together, mix well using pipette.
  
• French press; use the full cell holding (10 mL - 35 mL), and use 1500 psi pressure.
  
• Collect and reintroduce into french press 1x.
  
• Heat denature at 70 C for about 15 mins.
  
• Spin down, 10,000 x g for 30 mins.
  
• Collect and keep supernatant (SN) for IMAC purifications.
  
• Syringe filter the SN.
  

Immobilized metal ion affinity chromatography (IMAC) purification: (note: save portions at each step for protein gel).

• Prepare a 1 mL Ni-NTA resin column.
  
• Saturate column with 5x the column volume (so 5 mL) of lysis buffer. Repeat this step twice.
  
• Bind with 1:1 lysis buffer:SN sample.
  
• Wash with 1x column volume of lysis buffer.
  
• Wash with 5x (column volume) of wash buffer.
  
• Elute with 3 mL of elution buffer and collect all 3 mL of elution.
  

Dialysis: Link to dialysis cassette use

• Place dialysis cassette into storage buffer for 2 mins.
  
• Remove top and load dialysis cassette using a pipette or syringe.
  
• Squeeze the membrane to remove excess air.
  
• 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.
  
• Allow to sit for 2 - 4 hours.
  
• 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.
  
• Store at -20 C.
  

Validation

Protocol for harvesting phusion protein.

Materials needed:

• 50 mM NaH2PO4
  
• 300 mM NaCl
  
• 10mM Imidazole
  
• Adjust pH to 8.0 using NaOH
  

Wash Buffer:

• 50 mM NaH2PO4
  
• 300 mM NaCl
  
• 40mM Imidazole
  
• Adjust pH to 8.0 using NaOH
  

Elution Buffer:

• 50 mM NaH2PO4
  
• 300 mM NaCl
  
• 250 mM Imidazole
  
• Adjust pH to 8.0 using NaOH
  

Polymerase storage buffer: Make 3-4 Liters

• 50% Glycerol
  
• 100 mM Tris/HCl pH 8.0
  
• 0.2 mM EDTA
  
• 2 mM DTT
  
• 0.2% NP-40; nonionic detergent
  
• 0.2% Tween20
  

Procedure: (for 2 x 500 mL cultures)

• Grow overnight at 37 C shaking at 250 rpm.
  

• 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.
  

• Resuspend each cell pellet with 3 mL of lysis buffer and combine tubes together, mix well using pipette.
  
• French press; use the full cell holding (10 mL - 35 mL), and use 1500 psi pressure.
  
• Collect and reintroduce into french press 1x.
  
• Heat denature at 70 C for about 15 mins.
  
• Spin down, 10,000 x g for 30 mins.
  
• Collect and keep supernatant (SN) for IMAC purifications.
  
• Syringe filter the SN.
  

Immobilized metal ion affinity chromatography (IMAC) purification: (note: save portions at each step for protein gel).

• Prepare a 1 mL Ni-NTA resin column.
  
• Saturate column with 5x the column volume (so 5 mL) of lysis buffer. Repeat this step twice.
  
• Bind with 1:1 lysis buffer:SN sample.
  
• Wash with 1x column volume of lysis buffer.
  
• Wash with 5x (column volume) of wash buffer.
  
• Elute with 3 mL of elution buffer and collect all 3 mL of elution.
  

Dialysis: Link to dialysis cassette use

• Place dialysis cassette into storage buffer for 2 mins.
  

• Squeeze the membrane to remove excess air.
  
• 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.
  
• Allow to sit for 2 - 4 hours.
  
• 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.
  
• Store at -20 C.
  

Validation

Protocol for harvesting phusion protein.

Materials needed:

Glycerol stock of transformed E. coli cells expressing the His-tagged protein of interest.
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. link to strain

LB medium. LB recipe
Chloramphenicol stock. Cam recipe
Kanamycin stock. Kan recipe
Refrigerated centrifuge.

• 50 mM NaH2PO4
  
• 300 mM NaCl
  
• 10mM Imidazole
  
• Adjust pH to 8.0 using NaOH
  

Wash Buffer:

• 50 mM NaH2PO4
  
• 300 mM NaCl
  

• Adjust pH to 8.0 using NaOH
  

Elution Buffer:

• 50 mM NaH2PO4
  
• 300 mM NaCl
  
• 250 mM Imidazole
  
• Adjust pH to 8.0 using NaOH
  

• 50% Glycerol
  
• 100 mM Tris/HCl pH 8.0
  
• 0.2 mM EDTA
  
• 2 mM DTT
  
• 0.2% NP-40; nonionic detergent
  
• 0.2% Tween20
  

• Collect cells by centrifugation. Conditions as follows: 4 C, at 10,000 x g for 15 mins.
  

• French press; use the full cell holding (10 mL - 35 mL), and use 1500 psi pressure.
  

• Collect and keep supernatant (SN) for IMAC purifications.
  

• Prepare a 1 mL Ni-NTA resin column.
  

• Bind with 1:1 lysis buffer:SN sample.
  

Protocol for harvesting phusion protein.

Materials needed:

Glycerol stock of transformed E. coli cells expressing the His-tagged protein of interest.
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. link to strain

LB medium. LB recipe
Chloramphenicol stock. Cam recipe
Kanamycin stock. Kan recipe
Refrigerated centrifuge.
FRENCH laboratory press.
IPTG, 100 mM stock. Dissolve 2.38 g IPTG in 100 mL deionized water. Filter sterilize and store at -20 C.
Ni-NTA resin spin column. ThermoFisher HiPur Ni-NTA spin columns, 1ml (cat # 88225).
30k cutoff Amicon ultra-filtration membrane. Amicon Ultra-15 centrifugel filter units from Millipore (cat # UFC903008).

Lysis Buffer:

• 50 mM NaH2PO4
  
• 300 mM NaCl
  
• 10mM Imidazole
  
• Adjust pH to 8.0 using NaOH
  

Wash Buffer:

• 50 mM NaH2PO4
  
• 300 mM NaCl
  
• 20mM Imidazole
  
• Adjust pH to 8.0 using NaOH
  

Elution Buffer:

• 50 mM NaH2PO4
  
• 300 mM NaCl
  
• 250 mM Imidazole
  
• Adjust pH to 8.0 using NaOH
  

Polymerase storage buffer:

• 50% Glycerol
  
• 100 mM Tris/HCl pH 8.0
  
• 0.2 mM EDTA
  
• 2 mM DTT
  
• 0.2% NP-40; nonionic detergent
  
• 0.2% Tween20
  

Procedure: (for 2 x 500 mL culture)

• Streak LB plate supplemented with Kan and Cam using frozen stock. Growth overnight at 37 C.
  
• Inoculate 1.5 mL of LB broth supplemented with Kan and Cam with single colony from streak plate.
  
• Grow overnight at 37 C shaking at 200 rpm (????).
  
• 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.
  
• 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.
  
• Collect cells by centrifugation. Conditions as follows: 4 C, at 10,000 x g for 15 mins.
  
• Resuspend with 3 mL of lysis buffer and place tubes in cold room bath shaker (at dial 6 ?????) for ~ 2 hours.
  
• French press; use the full cell holding (10 mL - 35 mL), and use 1500 psi pressure.
  
• Lyse cells, heat denature at 70 C for about 15 mins.
  
• Spin down, 3700 - 4000 x g for 15 mins.
  
• Collect and keep supernatant (SN) for IMAC purifications.
  

IMAC purification:

• Prepare a 1 mL Ni-NTA resin column.
  
• Saturate column with 5x lysis buffer.
  
• Bind with 1:1 lysis buffer:SN sample.
  
• Wash with 5x wash buffer.
  
• Elute with elution buffer and collect samples after the first 1 mL flow through.
  
• Use 30k cutoff Amicon ultra-filtration membrane (???how do you use this???).
  
• Top-off with storage buffer.
  
• Spin at 4000 x g for 2.5 hours or so until volume is close to ~ 1 mL.