Transforming Electrocompetent Competent Cells

Adapted from Molecular Cloning: A Laboratory Manual 3rd Ed., Sambrook and Russell (2001)

SUPPLIES:

Equipment:

  • Shaking Incubator or Shaking Platform and 1L flask clamps
  • 42C Heating Bath or Block
  • 37C Incubator

Consumables:

  • Agar plates with appropriate antibiotic

Buffers and Solutions:

PROTOCOL:

  1. ) Remove one tube of cells from the freezer for each transformation reaction, plus appropriate controls
  2. ) Quickly thaw tubes by holding in your hand for a few seconds
  3. ) Add transforming DNA (up to 25ng, or 0.5-1uL of a miniprepped plasmid) and incubate on ice 30 min.
    • For transforming cloning reactions (e.g. from Gibson assembly or MegaWhop), it is possible to add up to 5μL of reaction directly to the cells without any purification steps
  4. ) Transfer tubes to a 42C heat block or water bath for EXACTLY 90 seconds
    • If using a heat block, make sure to fill the wells with diH2O to ensure efficient heat transfer
  5. ) Immediately transfer tubes to ice, cool 1-2 minutes
  6. ) Add 450μL SOC medium to each tube, and shake @200-250RPM for 45min
    • For tranformations in eppendorf tubes, can just put the tubes inside an empty 250mL flask and incubate in the large shaking incubators
  7. ) Plate at least two different volumes (10μL and 50μL usually work well) of each reaction on appropriate antibiotic plates

  • Note that this protocol can also be done in PCR plates for large numbers of transformations. Aliquot 10μL of cells in each well (this can be done when cells are prepared or with freshly-thawed cells) and add 0.5-1μL of DNA. Incubation steps can be done on the block of a pre-heated or -cooled thermocycler (timing is more exact if you heat the block first and transfer the plate by hand rather than changing temp of the block).

Electrocompetent E. coli

Making Electrocompetent E. coli Cells (small batch)

This procedure makes enough electrocompetent cells for 2-3 transformations.

  1. Grow an overnight culture of each strain in LB medium.
  2. Prepare 10 ml of fresh LB medium in a 50 ml flask for each strain.
  3. Inoculate with 100 ul of the overnight, stationary-phase culture.
    • In general, inoculate to OD600 of ~0.05.
  4. Grow the cells for approximately 2-3 hours, until they reach mid-exponential phase.
    • In general, this is an OD600 of ~0.6.
  5. Transfer the cells to 15 ml Falcon conical tubes.
  6. Pellet the cells by centrifugation for 5 minutes at 6,000 RPM. Remove promptly and pour off supernatant.
  7. Wash by adding 10 ml of chilled 10% glycerol to each tube, then vortexing vigorously to resuspend the pellet. Centrifuge for 3.5 minutes. Remove promptly and pour off supernatant. Repeat for at least four wash cycles in 10% glycerol.
  8. Resuspend in approximately 100 µl of 10% glycerol to make a 100x concentration of the initial culture.
  9. Divide into 30-50 µl aliquots in 0.5 or 1.7 ml tubes. Freeze or proceed directly to electroporation. Protocol for Electroporation of your Electrocompetent Cells can be found here.

Notes

  1. Keeping the cells cold during all processing steps is recommended. Ideally, chill the subculture in an ice water bath for >10 min before centrifuging, use a refrigerated centrifuge or a centrifuge in a cold room, and aliquot the final cell slurry into pre-chilled tubes for storage at -80C. These precautions are not strictly necessary if using the cells for routine cloning.
  2. Scale up the culture volumes if more electrocompetent cells are needed. The volume of 10% glycerol used for each wash should be at least 20— the volume of the cell pellet.

Making Electrocompetent E. coli Cells (large batch)

  1. Grow an overnight culture of each strain in LB medium.
  2. Prepare 100 ml of fresh LB medium in a 500 ml flask for each strain.
  3. Inoculate with 1 mL of the overnight, stationary-phase culture.
    • In general, inoculate to OD600 of ~0.05.
  4. Grow the cells for approximately 3-4 hours, until they reach mid-exponential phase.
    • In general, this is an OD600 of ~0.6.
  5. Transfer the cells to 2x 50 ml Falcon conical tubes.
  6. Pellet the cells by centrifugation for 5 minutes at 6,000 RPM. Remove promptly and pour off supernatant.
  7. Wash by adding 40 ml of chilled 10% glycerol to each tube, then vortexing vigorously to resuspend the pellet. Centrifuge for 3.5 minutes. Remove promptly and pour off supernatant. Repeat for at least four wash cycles in 10% glycerol.
  8. Resuspend each pellet in approximately 500 µl of 10% glycerol to make a 100x concentration of the initial culture.
  9. Divide into 30-50 µl aliquots in 0.5 or 1.7 ml tubes. Freeze promptly at -80šC.

-- Main.PengGeng - 25 May 2017

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Topic revision: r2 - 26 May 2017 - 01:04:38 - Main.PengGeng
 
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