Barrick Lab :: ProtocolsTestingTransformEff

---+ 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 / &mu;L 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 / &mu;g
   1 Expected efficiency for chemically competent cells prepared by the Inoue protocol is 1-3 x 10<sup>8</sup> colonies / &mu;g 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.

Barrick Lab > ProtocolList > ProtocolsTestingTransformEff

Contributors to this topic

KateElston, JeffreyBarrick

Topic revision: r3 - 2021-07-05 - 20:39:06 - Main.JeffreyBarrick