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Modified BL21 expression protocol

biotech , protocol

This is a modified protocol for transfecting DNA into BL21 E. coli or other strains of E. coli. It is based off of this protocol from the Thought Emporium, but with a few minor changes such as replacing LB broth media with SOC media for recovery after transfection.

Materials

  • distilled water

  • sterile 15ml tubes

  • sterile 1.5ml tubes

  • calcium chloride

  • syringe + syringe filter

  • LB broth

  • LB agar plate + appropriate antibiotic

  • S.O.C media

  • Innoculation loop

  • Sterile spreader

  • Pipettes/tips

  • 15ml centrifuge

  • 1.5ml centrifuge

  • heat block

  • Ice bath

  • shaking/rotating incubator

Stock prep

  1. Prepare a 1M calcium chloride solution by dissolving 1.1g in 10ml of water

  2. transfer 1ml of this solution to a fresh tube and add 9ml of distilled water

  3. filter sterilize into a fresh tube.

  4. Label the result “transformation buffer”

  5. For improved results, prechill this buffer in the fridge for at least an hour before use

Day 1

  1. The day before performing the transformation protocol, innoculate 10ml of LB broth in a 15ml tube with BL21 cells, or any other ecoli variety

  2. place into rotating/shaking incubator at 37C and let grow overnight

Day 2

  1. Innoculate 10ml of fresh LB with 100ul of overnight solution
  • Let grow for exactly 2H (do not let overgrow)

  1. Pellet cells by centrifugation at 5000rpm for 2-3 minutes

  2. Decant off liquid and resuspend pellet in 1ml transformation buffer

  3. Transfer liquid to 1.5ml tube

  4. Pellet cells at 10000rpm for 45 - 60 seconds

  5. Pipette off liquid, do not disutrb pellet

  6. Add 1ml of transformation buffer and resuspend by pipetting gently

  7. Pellet again at 10000rpm for 45 - 60 seconds

  8. Pipette off liquid

  9. The pellet should contain enough cells for 3-4 reactions. Add 100ul of transformation buffer for the number of reactions you wish to perform (100-400ul)

  • If doing more than 1 reaction, seperate liquid into 100ul aliqotes in seperate tubes

  1. Add 50-400ng of DNA to each tube (50 for easy plasmids/strains, 400 for stubborn ones. Adjust as needed)

  2. incubate mixutre in a fridge (5C) for 25 min

  3. Preheat hot plate/water bath to 42C

  4. After incubation in the fridge heat shock cells in heat block for exactly 90s

  5. immeditaly chill in ice bath after heat shocking for 3-5 min

  6. Add 1ml of fresh S.O.C media to each tube, and allow to recover in an incubator at 37C for 1-2H with shaking/rotation (if S.O.C media is not available. This can be substituted with LB broth, but will result in lower transformation efficiency)

If using turbo or other easy strains:

  1. plate 100ul of recovered solution on appropriate antibiotic or selection plates

  2. spread using sterile spreader or innoculation loops

If using BL21:

  1. Pellet cells by centrifugation at 10000rpm for 20s

  2. plate entire pellet + 100ul of media

  3. spread using sterile spreader or innoculation loop

  1. Incubate plate at 37C for 2-3 days until colonies develop