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Finkelstein Lab Wiki

3xFLAG Lac1

Used in our papers:12

Lac1 repressor with 3xFLAG epitope at the C-terminus

Contents

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

1. Transform

Section titled “1. Transform”
  1. Transform 1-2 uL of 3xFLAG LacI plasmid into 50 uL of BL21(Arctic Express) cells.
  2. Flick 5 times.
  3. Place on ice for 5 minutes.
  4. Place in a 42C water bath for 30 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 37C, 250 rpm.
  7. Plate 150 uL of cells onto an LB-Carb plate and place in a 37C incubator for 24 hours.
  8. You can hold plate for 2 weeks in 4C 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 a 50 ug/mL concentration of carbenicillin.
  2. Grow the flask overnight for 12-18 hours.
  3. In the morning, inoculate 6L of LB with 15 mL of overnight per liter.
    • Each liter should have a carbenicillin concentration of 50 ug/mL
  4. Grow @ 37C until an O.D. 600 of 0.6-1.0.
  5. Once the O.D. 600 is reached, inoculate the cultures with a concentration of 2 mM IPTG.
  6. Grow 2-3 @ 37C.
  7. Harvest by centrifuging for 10 minutes at 5000 rpm (@ 4C).
  8. Decant the supernatant and keep the pellet Do this in the cold room.
  9. 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)
  10. Flash freeze cells in liquid nitrogen and store in -80C 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 4C.
  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) (Save 20 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 20 mL of cleavage buffer to the chitin beads and allow 10 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. Kim et al. (2017). Efficient modification of λ-DNA substrates for single-molecule studies. Sci Rep. ↩

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