IGEM:Virginia 2012/Protocols/CaCl2 Competent Stock Cells: Difference between revisions
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==Overview== | ==Overview== | ||
Detailed below is the procedure to make CaCl<sub>2</sub>-competent stock cells. | 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== | ||
Revision as of 07:30, 4 June 2012
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 .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
Contact
- Who has experience with this protocol?
or instead, discuss this protocol.
