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Finkelstein Lab Wiki

EcoRI(E111Q-3xHA) Purification

Used in our papers:12

Contents

Section titled “Contents”
  1. Transform
  2. Express
  3. Purify
  4. Storage

1. Transform

Section titled “1. Transform”
  1. Thaw BL21 cells on ice for 20 minutes.
  2. Transform 1-2 µL of plasmid into 50 µL of BL21(Arctic Express) cells.
  3. Flick 5 times.
  4. Place on ice for 5 minutes.
  5. Place in a 42°C water bath for 30 seconds.
  6. Place on ice for 2 minutes.
  7. Inject the cells into 500 µL of SOC and place in a shaking incubator for 1 hour at 37°C, 250 RPM.
  8. Plate 150 µL of cells onto an LB-antibiotic plate and place in a 37°C incubator for 24 hours.
  9. You can hold plate for 2 weeks in 4°C if you parafilm the edges.

2. Express

Section titled “2. Express”
  1. Take one cell from BL21(Arctic Express) plate and inoculate 50 mL of LB in a 500 mL flask with antibiotic.
  2. Grow the flask overnight for 12-18 hours.
  3. In the morning, inoculate 6L of LB with 15 mL of overnight per liter.
    1. Each liter should have antibiotic.
    2. Grow @ 37°C until log phase (OD600 of 0.6) is reached.
    3. Once in log phase, inoculate the cultures with a concentration of 2 mM IPTG.
    4. Grow 2-3 hours @ 37°C.
    5. Harvest by centrifuging for 10 minutes at 5000 rpm (@ 4°C).
    6. Decant the supernatant and keep the pellet. Do this in the cold room
    7. Resuspend each 3L pellet to 35 mL with resuspension buffer:
      • 50 mM Tris pH 7.5
      • 500 mM NaCl
      • 1 mM EDTA
      • 10% Sucrose
      • 1 mM PMSF (protease inhibitor)
    8. Flash freeze cells in liquid nitrogen and store in -80°C until ready to use.

3. Purify

Section titled “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).
  3. Centrifuge the lysate in the ultracentrifuge at 35K for 35 minutes at 4°C.
  4. Take a sample of the clarified lysate for later gel analysis.
  5. Load the clarified lysate onto a 10 mL pre-equilibrated chitin column.
    • Equilibration buffer (Buffer A):
      • 50 mM Tris pH 7.5
      • 100 mM NaCl
      • 1 mM EDTA
  6. Wash the column with 1L of Wash Buffer (Buffer B), minimum 500mL. (Save 40 mL to convert into cleavage buffer)
    • Wash Buffer:
      • 20 mM Tris pH 7.5
      • 1M NaCl
      • 1 mM EDTA
  7. Take the saved Buffer B and add a 50 mM Concentration of DTT (Cleavage Buffer).
  8. Add 40 mL of cleavage buffer to the chitin beads and allow 30 mL to drip through.
  9. Allow the other 10 mL to sit overnight.
  10. In the morning collect 1 mL fractions and analyze all fractions collected throughout the process.

4. Storage

Section titled “4. Storage”
  1. Dialyze into storage buffer:
    • 50 mM Tris pH 7.5
    • 200 mM NaCl
    • 1 mM EDTA
    • 50% Glycerol
  2. CHECK THE PH OF STORAGE BUFFER AND ADJUST TO 7.5
  3. Aliquot fractions into 10-20 uL in 500 uL Eppendorf tubes and flash freeze with liquid nitrogen.

Footnotes

Section titled “Footnotes”

  1. Brown et al. (2016). Dynamic DNA binding licenses a repair factor to bypass roadblocks in search of DNA lesions. Nature Communications. ↩

  2. Dillard et al. (2018). Assembly and Translocation of a CRISPR-Cas Primed Acquisition Complex. Cell. ↩