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
  • 42°C Heating Bath or Block
  • 37°C 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 µL of cells using the standard transformation protocol.
  2. Plate 50 µL 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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