Team:IIT Madras/Notebook/Protocols

ULTRACOMPETENT CELL PREPARATION

• Materials/Buffers
• SOB SOLUTION FOR COMPETENT CELL PREPARATION
1. 0.5% yeast Extract
2. 2% Tryptone
3. 10mM NaCl
4. 2.5mM KCl
5. 10mM MgCl2
6. 10mM MgSO4.
7. Dissolve all in nanopure water and autoclave
• TRANSFORMATION BUFFER FOR COMPETENT CELL PREPARATION
1. 10mM PIPES
2. 15mM CaCl2
3. 250mM KCl
4. 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.