---+ 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 [[ProtocolsChemCompCellsInoue][Inoue method]] or another method * 10pg / μL solution of a standard plasmid (e.g. pUC19) * [[ProtocolsRecipesSOB][SOC Medium]] ---++ PROTOCOL 1 Transform 10 pg of a standard plasmid (e.g. pUC19) in 50 µL of cells using the standard transformation protocol. 1 Plate 50 µL of transformed cells in triplicate on appropriate antibiotic 1 Grow plates overnight, and count colonies the next morning 1 Average colony counts for the three plates; efficiency = (average # colonies) x 10<sup>6</sup> cfu / μg 1 Expected efficiency for chemically competent cells prepared by the Inoue protocol is 1-3 x 10<sup>8</sup> colonies / μg of plasmid ---++ OTHER RESOURCES * [[http://parts.igem.org/Help:Competent_Cell_Test_Kit][iGEM protocol for testing chemically competent cells]] – describes a similar procedure in more depth. * [[https://www.neb.com/products/c2987-neb-5-alpha-competent-e-coli-high-efficiency#Product%20Information][NEBalpha competent cells]] – has some tables showing the effects of changing the amount of plasmid transformed and various other parameters.
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Topic revision: r3 - 2021-07-05 - JeffreyBarrick