IGEM:Virginia 2012/Protocols/CaCl2 Competent Stock Cells

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Current revision (11:35, 4 June 2012) (view source)
 
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==Overview==
==Overview==
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Detailed below is the procedure to make CaCl<sub>2</sub>-competent stock cells.
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Detailed below is the procedure to make CaCl<sub>2</sub>-competent stock cells. This is adapted from [http://sites.bio.indiana.edu/~chenlab/potocols/CompetentCellStock.htm The Department of Biology at Indiana University].
==Materials==
==Materials==
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* Pick single colony and grown in 5 mL LB media for 2-3 hrs with vigorous shaking (~300rpm).
* Pick single colony and grown in 5 mL LB media for 2-3 hrs with vigorous shaking (~300rpm).
   
   
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* Transfer into 200 mL LB media and grow cells with vigorous shaking until an absorbance of .6A600 is achieved; put on ice and keep chilled at 0ºC for 15 min.
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* Transfer into 200 mL LB media and grow cells with vigorous shaking until an absorbance of A600=0.6 is achieved; put on ice and keep chilled at 0ºC for 15 min.
   
   
* Apply cells into four 50 mL sterile centrifuge (conical) tubes and spin cells at 4000 rpm for 10 min.
* Apply cells into four 50 mL sterile centrifuge (conical) tubes and spin cells at 4000 rpm for 10 min.
   
   
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* Resuspend cells with 15 mL sterile, ice-cold .1M CaCl2 by gentle pipetting (DO NOT VORTEX).  Leave on ice 15 min.  Spin again at 4000 rpm for 10 min.
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* Resuspend cells with 15 mL sterile, ice-cold 0.1M CaCl2 by gentle pipetting (DO NOT VORTEX).  Leave on ice 15 min.  Spin again at 4000 rpm for 10 min.
   
   
* Resuspend in 4 mL sterile, ice-cold 0.1 M CaCl2 /15% glycerol (DO NOT VORTEX).
* Resuspend in 4 mL sterile, ice-cold 0.1 M CaCl2 /15% glycerol (DO NOT VORTEX).
   
   
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**Optional: Leave on ice 4 to 21 hours (BL21 for 4 hours, DHa and XL-1 blue for 0/n).
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*Optional: Leave on ice 4 to 21 hours (BL21 for 4 hours, DHa and XL-1 blue for 0/n).
   
   
* Freeze aliquots of cells in sterile and labeled microcentrifuge tubes.
* Freeze aliquots of cells in sterile and labeled microcentrifuge tubes.

Current revision

Contents

Overview

Detailed below is the procedure to make CaCl2-competent stock cells. This is adapted from The Department of Biology at Indiana University.

Materials

  • 5 mL LB Media
  • 200 mL LB Media
  • Ice
  • Four 50 mL sterile centrifuge (conical) tubes
  • Centrifuge
  • 0.1 M CaCl2
  • 0.1 M CaCl2 / 15% glycerol

Procedure

Day 1: Plate cells and grow overnight.

Day 2: Entire procedure should take approximately one hour.

  • Pick single colony and grown in 5 mL LB media for 2-3 hrs with vigorous shaking (~300rpm).
  • Transfer into 200 mL LB media and grow cells with vigorous shaking until an absorbance of A600=0.6 is achieved; put on ice and keep chilled at 0ºC for 15 min.
  • Apply cells into four 50 mL sterile centrifuge (conical) tubes and spin cells at 4000 rpm for 10 min.
  • Resuspend cells with 15 mL sterile, ice-cold 0.1M CaCl2 by gentle pipetting (DO NOT VORTEX). Leave on ice 15 min. Spin again at 4000 rpm for 10 min.
  • Resuspend in 4 mL sterile, ice-cold 0.1 M CaCl2 /15% glycerol (DO NOT VORTEX).
*Optional: Leave on ice 4 to 21 hours (BL21 for 4 hours, DHa and XL-1 blue for 0/n).

  • Freeze aliquots of cells in sterile and labeled microcentrifuge tubes.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!


References

Contact

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