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Team:IIT Madras/Notebook/Protocols
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Revision as of 06:12, 18 October 2009
IIT Madras
ULTRACOMPETENT CELL PREPARATION
- Materials/Buffers
- SOB SOLUTION FOR COMPETENT CELL PREPARATION
- 0.5% yeast Extract
- 2% Tryptone
- 10mM NaCl
- 2.5mM KCl
- 10mM MgCl2
- 10mM MgSO4.
- Dissolve all in nanopure water and autoclave
- TRANSFORMATION BUFFER FOR COMPETENT CELL PREPARATION
- 10mM PIPES
- 15mM CaCl2
- 250mM KCl
- Dissolve in nanopure water and adjust pH to 6.7 with KOH or HCl. The add MnCl2 to 55mM and adjust final volume. Sterilize by filtration with 0.45 µ filter. Store at 40C
- Cells were cultured on LB agar plate overnight at 370C.
- 10-12 colonies were cultured in 250ml SOB medium.
- It was incubated at 370C for 1hour. Then the flasks were transferred to 190C. It was incubated toll OD600 reached 0.5
- Flask was placed in ice for 10min.
- The cells were pelleted by spinning at 4000rpm for 10min at 40C.
- Cells were resuspended in 80ml ice cold TB(Transformation Buffer) and stored on ice for 10min.
- It was centrifuged again at 4000rpm for 10min at 40C.
- Pellet was resuspended in 20ml of TB with 1.5ml DMSO.
- 100-500µl of the cells were aliquoted and stored at -800C.
