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 or another method
  • 10pg / μL solution of a standard plasmid (e.g. pUC19)
  • SOC Medium

PROTOCOL

  1. Transform 10 pg of a standard plasmid (e.g. pUC19) in 50 L of cells using the standard transformation protocol.
  2. Plate 50 L 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 = (average # colonies) x 106 cfu / μg
  5. Expected efficiency for chemically competent cells prepared by the Inoue protocol is 1-3 x 108 colonies / μg of plasmid

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Contributors to this topic Edit topic KateElston, JeffreyBarrick
Topic revision: r3 - 2021-07-05 - 20:39:06 - Main.JeffreyBarrick
 
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