IGEM:Virginia 2012/Protocols/CaCl2 Competent Stock Cells: Difference between revisions
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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== | ||
Detailed below is the procedure to make CaCl<sub>2</sub>-competent stock cells. | |||
==Materials== | ==Materials== | ||
* | * 5 mL LB Media | ||
* | * 200 mL LB Media | ||
* | * Ice | ||
* | * Four 50 mL sterile centrifuge (conical) tubes | ||
* Centrifuge | * Centrifuge | ||
* 0. | * 0.1 M CaCl<sub>2</sub> | ||
* | * 0.1 M CaCl<sub>2</sub> / 15% glycerol | ||
==Procedure== | ==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). | ||
* | |||
* Resuspend | * 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== | ==Notes== |
Revision as of 07:29, 4 June 2012
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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