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Tutrorial

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PREPARATION OF COMPETENT CELLS FOR ELECTROPORATION1

You will use your ligated DNA to transform XL1-Blue cells ® (Stratagene), a strain of E.coli that is tetracycline resistant and is easily transformed by electroporation. For use in electroporation the cells must be prepared in a medium with a minimal amount of salt. Why? We will also prepare them in a solution that contains glycerol so that they can be stored at -80oC and then thawed without lose of integrity of the cell membranes. The procedure outlined below follows the protocol provided by Eppendorf with the Electroporator 2510. The first two steps of the procedure have been done for you.
  1. Start a fresh overnight culture with XL1-Blue cells. Grow at 37oC with moderate shaking.
  2. Inoculate 1 liter of Luria Broth with 5 ml (1:200) of the fresh O/N culture, being sure that the flask is large enough to allow adequate aeration. Grow at 37oC with vigorous shaking to an OD600 of 0.5 to 0.6.
  3. Chill the cells in an ice-water bath for 15 minutes and transfer to a pre-chilled centrifuge bottle. Centrifuge for 20 minutes at 5000x g, 2-4oC. Re-suspend the pellet in 1 liter of ice-cold sterile water. It is important to keep the cells cold during this entire procedure.
  4. Centrifuge as above. Repeat the wash with cold sterile water. Pour off the supernatant immediately and re-suspend the pellet by swirling in the remaining liquid.
  5. Add 40 ml of ice-cold 10% glycerol, mix and centrifuge 10 min at 5000x g, 2-4oC. Remove the supernatant. Estimate the pellet volume, add an equal volume of ice-cold10% glycerol to re-suspend the cells and aliquot 40 µl to pre-chilled 0.6 ml microcentrifuge tubes. Quick-freeze in liquid nitrogen and store at -80oC.

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1 You may also transform the cells by heat shock. However, the transformation efficiency of heat shock is much lower than that of electroporation. Protocols for heat shock transformation and preparation of cells for heat shock transformation can be found in Sambrook et al. (2001). A protocol is also included with this set of Procedures.