ProtocolsColonyTransformation < Lab

---+ Colony Transformation

*%RED%This is a theoretical protocol that has not been tested!%ENDCOLOR%*

This protocol and be used for the rapid preparation of chemically competent _E. coli_ cells from colonies on a plate for transformation of a high copy number plasmid. The efficiency of this protocol has been reported as 5 x 10<sup>3</sup> to 5 x 10<sup>4</sup> colonies per microgram of plasmid. This is 2-200 fold less than for normal chemically competent cells. Thus, it should only be used for an existing plasmid (not a new ligation or cloning product where an even lower efficiency is expected).

---++ Materials
   * 50 mM sterilized CaCl solution
   * LB-antibiotic plate
   * Ice bucket 
   * 42°C water bath

---++ Procedure
   1 Add 250 µl of CaCl<sub>2</sub> for each transformation to a test tube. Place on ice.
   1 Transfer one (or more) large colonies from the agar plate to each test tube using an inoculating loop.
      * You are aiming for ~10-25 million cells
      * %X% Be careful to not transfer any agar
   1 Resuspend the cells in each test tube by pipetting up and down with a P1000.
   1 Add plasmid to each tube and incubate for 15 minutes on ice.
   1 Heat shock cells by moving tubes to 42°C for 30-90 seconds.
      * Time of heat shock depends on thermal transfer properties of the test tube.
   1 Immediately move tubes back to ice for at least 1 minute.
   1 Add 250 µl of LB to each tube.
   1 Incubate for 5-30 minutes shaking at 37°C if needed for antibiotic marker.
   1 Plate 100 µl from each tube on prewarmed LB plates.
      * Plate 40 µl of a 10<sup>4</sup> dilution of the LB culture on a plate *without* antibiotic in order to estimate the transformation efficiency. You should get ~80-200 cells depending on the size of your colony.

*Animated protocol*:
   * [[http://www.cellsystems.org/education/ref/BHS/contrans/contrans01.html][Animated Protocol – Cell Systems Initiative (UW Bioengineering)]]

---++ References
   * [[http://labprotocols.dnalc.org/files/039_bacterial_transformation.pdf][DNA Lab Protocols]]
Barrick Lab > ProtocolList > ProtocolsColonyTransformation
Topic revision: r1 - 2016-02-18 - 22:29:09 - Main.JeffreyBarrick