Checking Transformation Efficiency of 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
  • Colony Counter

Consumables:

  • Agar plates with appropriate antibiotic

Buffers and Solutions:

  • Competent cells prepared using the Inoue method
  • 10pg / μL solution of a standard plasmid (e.g. pUC19)
  • SOC Medium

PROTOCOL:

  1. ) Transform 10pg of a standard plasmid (e.g. pUC19) in 50μL of cells using the standard transformation protocol
  2. ) Plate 50uL of transformed cells in triplicate on appropriate antibiotic
  3. ) Grow plates overnight, and count colonies the next morning
  4. ) Average colony counts for the three plates; efficiency = (# colonies) x 106 cfu / μg
  5. ) Expected efficiency for the Inoue protocol is 1-3 x 108 colonies / μg of plasmid

-- Main.ColinBrown - 15 Dec 2016


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Topic revision: r1 - 23 Feb 2017 - 21:41:22 - Main.ColinBrown