Transforming Chemically 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).

-- Main.ColinBrown - 15 Dec 2016


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Topic revision: r1 - 23 Feb 2017 - 21:38:46 - Main.ColinBrown