Barrick Lab :: ProtocolsChemCompCellsHub

---+ Chemically Competent Cells


---++ Preparation of Chemically Competent Cells

There are a few variations on the protocol for preparation of chemically competent cells. Choice of protocol may depend on amount of time involved in the prep and the transformation efficiency of the prepared cells.

   * [[ProtocolsChemCompCellsCaCl2Glycerol][CaCl<sub>2</sub>/Glycerol method]] - "quick and efficient" method
   * [[ProtocolsChemCompCellsInoue][No-Kit Inoue Chemically Competent Cells]] 

After preparation, the transformation efficiency of the chemically competent cells should be evaluated by [[ProtocolsTestingTransformEff][transforming them with a standard plasmid]].
 

---++ Transforming 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

---++++ Consumables:

   * Agar plates with appropriate antibiotic

---++++ Buffers and Solutions:

   * Competent Cells prepared using one of the methods listed above
   * [[ProtocolsRecipesSOB][SOC Medium]]

---++ PROTOCOL:

   1 ) Remove one tube of cells from the freezer for each transformation reaction, plus appropriate controls
   1 ) Thaw tubes on ice
   1 ) Add transforming DNA (up to 25ng, or 0.5-2&mu;L of a miniprepped plasmid) and incubate on ice 30 min. 
      * For transforming cloning reactions (e.g. from Gibson assembly or MegaWhop), it is possible to add up to 5&mu;L of reaction directly to the cells without any purification steps
   1 ) Transfer tubes to a 42°C heat block or water bath for *EXACTLY* 90 seconds 
      * If using a heat block, make sure to fill the wells with diH<sub>2</sub>O to ensure efficient heat transfer
   1 ) Immediately transfer tubes to ice, cool 1-2 minutes
   1 ) Add 450&mu;L SOC medium to each tube, and shake @ 200-250RPM for 1 hour 
      * For tranformations in eppendorf tubes, can just put the tubes inside an empty 250mL flask and incubate in the large shaking incubators
   1 ) Plate at least two different volumes (10&mu;L and 50&mu;L usually work well) of each reaction on appropriate antibiotic plates
      * If your efficiencies tend to be on the low side, pellet your cells by centrifuging for 5 min at 3000rpm then resuspend in 100&mu;L and plate the entire mixture

   * Note that this protocol can also be done in PCR plates for large numbers of transformations. Aliquot 10&mu;L of cells in each well (this can be done when cells are prepared or with freshly-thawed cells) and add 0.5-1&mu;L of DNA. Incubation steps can be done on the block of a pre-heated or -cooled thermocycler (timing is more exact if you heat the block first and transfer the plate by hand rather than changing temp of the block).

---+++ Protocol Contributors:
   * Colin Brown
   * Matt McGuffie
   * Kate Elston
Barrick Lab > ProtocolList > ProtocolsChemCompCellsHub
Contributors to this topic
KateElston, JeffreyBarrick, CameronRoots, AlexaMorton
Topic revision: r5 - 2025-03-20 - 19:33:18 - Main.CameronRoots