CONTROLLER Chemically Competent Cells

Heat Shock Transformation

Sigma CONTROLLER Chemically Competent Cells are provided in 40 μL aliquots, sufficient for a single transformation. Transformation is performed by incubation on ice followed by heat shock at 42°C. For maximal transformation efficiency, the heat shock is performed in 15 mL polypropylene culture tubes (17 x 100 mm). The use of other types of tubes may dramatically reduce the transformation efficiency. To ensure successful transformation results, the following precautions must be taken:

  • All culture tubes must be thoroughly pre-chilled on ice before use.
  • The cells must be completely thawed on ice before use.

As a control for transformation, CONTROLLER competent cells are provided with a vial of pUC19 DNA at a concentration of 10 pg/μL. Use 1 μL (10 pg) for transformation. Select pUC19 transformants on plates containing ampicillin (100 μg/mL).

Transformation Protocol

  1. Remove Recovery Medium from the freezer and bring to room temperature.
  2. Remove HI-Control cells from the -80°C freezer and thaw completely on wet ice (10-15 minutes).
  3. Briefly centrifuge thawed plasmid DNA to collect the solution in the bottom of the tube.
  4. Transfer the cells to a pre-chilled disposable polypropylene 15 mL culture tube (17 x 100 mm).
  5. Add 1 μL of DNA (10 pg to 100 ng) to the cells. Stir briefly with pipet tip. Do not pipet up and down to mix, which can introduce air bubbles and warm the cells.
  6. Incubate culture tube containing cells and DNA on ice for 30 minutes.
  7. Heat shock cells by placing the tube in a 42°C water bath for 45 seconds.
  8. Return the tube of cells to ice for 2 minutes.
  9. Immediately following the 2 minute ice incubation, add 960 μL of room temperature Recovery Medium to the cells in the culture tube.
  10. Place the tube in a shaking incubator at 250 rpm for 1 hour at 37°C.
  11. Plate 50 μL of transformed cells on LB agar plates containing the appropriate antibiotic. For pUC19 control transformations, CONTROLLER BL21(DE3) cells should yield >5 colonies, and CONTROLLER SIG10 cells should yield >500 colonies.
  12. Incubate the plates overnight at 37°C.

Materials

     
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