IGEM:Virginia 2012/Protocols/CaCl2 Competent Stock Cells

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(New page: ==Overview== This is how to isolate the phage genome from a preparation of bacteriophages, specifically the ''Bordetella'' phage. ==Materials== * 10 mM EDTA * CsCl-banded phage * steril...)
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==Overview==
==Overview==
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This is how to isolate the phage genome from a preparation of bacteriophages, specifically the ''Bordetella'' phage.
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Detailed below is the procedure to make CaCl<sub>2</sub>-competent stock cells.
==Materials==
==Materials==
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* 10 mM EDTA
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* 5 mL LB Media
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* CsCl-banded phage
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* 200 mL LB Media
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* sterile tube
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* Ice
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* 100% ethanol
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* Four 50 mL sterile centrifuge (conical) tubes
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* -20°C freezer
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* Centrifuge
* Centrifuge
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* 0.5 mL of 70% ethanol
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* 0.1 M CaCl<sub>2</sub>
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* TE Buffer
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* 0.1 M CaCl<sub>2</sub> / 15% glycerol
==Procedure==
==Procedure==
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* Add 10 volumes of 10mM EDTA pH 8.0 to 1 volume of CsCl-banded phage in a sterile tube
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Day 1: Plate cells and grow overnight.
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* Heat for 5 minutes at 65°C; let cool to room temperature
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* Add 2.5 volumes 100% ethanol; place in -20°C freezer for 20 minutes
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Day 2:  Entire procedure should take approximately one hour.
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* Centrifuge: 10,000g for 5 minutes
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* Discard supernatant; rinse pellet with 0.5ml 70% ethanol; centrifuge again; discard supernatant; rinse pellet with 0.5ml 70% ethanol; centrifuge again; discard supernatant
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* Pick single colony and grown in 5 mL LB media for 2-3 hrs with vigorous shaking (~300rpm).
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* Let pellet air dry at room temperature
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* Resuspend DNA in TE buffer; let DNA sit for several hours to go into solution; or, restriction digests can be performed immediately; store solution of DNA at -20°C for long-term storage (i.e. months) or at 4°C for short-term storage (i.e. 1-2 days)
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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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* 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 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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* Freeze aliquots of cells in sterile and labeled microcentrifuge tubes.
==Notes==
==Notes==

Revision as of 09:29, 4 June 2012

Contents

Overview

Detailed below is the procedure to make CaCl2-competent stock cells.

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 .6A600 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 .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

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