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Φ29 (phi29) DNA Polymerase Purification

Used in our papers:123

phi29 DNA Polymerase is the replicative polymerase from the Bacillus subtilis phage phi29 (Φ29). This polymerase has exceptional strand displacement and processive synthesis properties. The polymerase has an inherent 3´→5’ proofreading exonuclease activity.

Contents

Section titled “Contents”
  1. Notes
  2. Purification Protocol
    1. Transform
    2. Express
    3. Purify
    4. Dialyze
    5. Analysis

1. Notes

Section titled “1. Notes”
  • Φ29 is 67KDa

2. Purification Protocol

Section titled “2. Purification Protocol”

2.1 Transform

Section titled “2.1 Transform”
  1. Transform 1 uL of ϕ29 (his/flag, his/tev, or his/tev/flag) plasmid into 50 uL of BL21(Arctic Express) cells.
  2. Flick 5 times.
  3. Place on ice for 5 minutes.
  4. Place in a 42oC water bath for 42 seconds.
  5. Place on ice for 2 minutes.
  6. Inject the cells into 500 uL of SOC and place in a shaking incubator for 1 hour at 37oC, 250 rpm.
  7. Plate 200 uL of cells onto an LB-Carb plate and place in a 37oC incubator for 24 hours.
  8. You can hold plate for 2 weeks in 4oC if you parafilm the edges.

2.2 Express

Section titled “2.2 Express”
  1. Take one cell from BL21(A.E.) plate and inoculate 100 mL of LB in a 500 mL flask with a 50 ug/mL concentration of carbenicillin.
  2. Grow the flask overnight for 12-18 hours (37oC).
  3. In the morning, inoculate 9L (6x 1.5 L) of LB with 15 mL of overnight per 1.5 liters.
  4. Each 1.5 liters should have a carbenicillin concentration of 50 ug/mL.
  5. Grow @ 30oC until an O.D. 600 of 0.6-1.0.
  6. Once the O.D. 600 is reached, inoculate the cultures with a concentration of 0.4 mM IPTG.
  7. Grow O/N @ 18oC (16-20 hours).
  8. Harvest by centrifuging for 10 minutes at 5000 rpm (4oC).
  9. Decant the supernatant. Keep pellet on ice and move to cold room ASAP
  10. Resuspend each 3L pellet to 35 mL with resuspension buffer (105 mL total):
    • 25 mM Tris pH 7.5
    • 500 mM NaCl
    • 5% Glycerol
    • 5 mM imidazole
    • Add Halt Protease Inhibitor Cocktail (No EDTA) to final [] of 1X. Also add 2mM benzamidine.
  11. Proceed directly to purification for (1) 3L pellet. Flash freeze two other pellets.

2.3 Purify

Section titled “2.3 Purify”
  1. Defrost the pellet on ice.
  2. Sonicate the lysate for a total of 90 seconds (15 second bursts with 90 second rests in between) on ice.
  3. Centrifuge the lysate in the ultracentrifuge at 35K for 35 minutes at 4oC.
  4. Take a sample of the clarified lysate for later gel analysis.
  5. Load the clarified lysate onto a 5 mL pre-equilibrated Ni column (HisTrap HP).
    • Equilibration buffer (Buffer A):
      • 25 mM Tris pH 7.5
      • 500 mM NaCl
      • 5% Glycerol
      • 5 mM imidazole
  6. Start collecting flow thru.
  7. Wash the column with Buffer A until abs comes down (indicating column is thoroughly washed).
  8. Elute with Elution buffer and collect 1mL fractions:
    • 25 mM Tris pH 7.5
    • 500 mM NaCl
    • 5% Glycerol
    • 300 mM imidazole
  9. Pool peak fractions as well as flanking fractions (2mL either side).
  10. Add elute to a 10 mL pre-equilibrated chitin column:
    • Equilibration buffer:
      • 25 mM Tris pH 7.5
      • 500 mM NaCl
      • 0.1 mM EDTA
  11. Wash with 1L of this Equilibration buffer (save 20 mL to convert into Cleavage buffer).
  12. Add DTT to a final concentration of 50 mM to the Cleavage buffer.
  13. Add 20 mL of cleavage buffer to the chitin beads and allow 10 mL to drip through Collect this 10 mL for gel
  14. Allow the other 10 mL to sit overnight.
  15. In the morning collect 1 mL fractions and analyze all fractions collected throughout the process on a 10% gel.

2.4 Dialyze

Section titled “2.4 Dialyze”
  1. Dialyze the pooled fractions into Storage buffer:
    • 10 mM Tris pH 7.5
    • 100 mM KCl
    • 1 mM DTT
    • 0.1 mM EDTA
    • 50% Glycerol
    • CHECK pH OF SOLUTION. ADDING GLYCEROL WITH DECREASE OVERALL pH. pH with NaOH if needed
  2. Aliquot fractions into 10-20 uL in 500 uL Eppendorf tubes and flash freeze with liquid nitrogen. Store in either -80 or - 20 freezer Samples will not “freeze” because of high glycerol.

2.5 Analysis

Section titled “2.5 Analysis”
  1. Analyze the protein using a Bradford Assay.
    • BSA curve and sample, incubate w/ dye for ~5 minutes (stay consistent with times)
  2. Analyze the protein on a 10% gel for concentration and degradation/contamination.
    1. Setup BSA standard.
    2. Boil samples for 10 minutes.
    3. Spin samples at 15000 rpm for 3 minutes.
    4. Load onto gel and run 150-180V (lower V = nicer runs/gels).
  3. Check the DNA to protein ratio (260/280) using the nanodrop.

Footnotes

Section titled “Footnotes”

  1. Gibb et al. (2012). Single-stranded DNA curtains for real-time single-molecule visualization of protein-nucleic acid interactions. Anal. Chem. ↩

  2. Gallardo et al. (2015). High-Throughput Universal DNA Curtain Arrays for Single-Molecule Fluorescence Imaging. Langmuir. ↩

  3. Soniat et al. (2017). Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination. Methods in Enzymology. ↩