From 2009.igem.org
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| - | PROTOCOLS
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| - | 1) ULTRACOMPETENT CELL PREPARATION
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| - | a. Materials/Buffers
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| - | i. SOB SOLUTION FOR COMPETENT CELL PREPARATION
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| - | 1. 0.5% yeast Extract
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| - | 2. 2% Tryptone
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| - | 3. 10mM NaCl
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| - | 4. 2.5mM KCl
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| - | 5. 10mM MgCl2
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| - | 6. 10mM MgSO4.
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| - | 7. Dissolve all in nanopure water and autoclave
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| - | ii. TRANSFORMATION BUFFER (TB) FOR COMPETENT CELL PREPARATION
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| - | 1. 10mM PIPES
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| - | 2. 15mM CaCl2
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| - | 3. 250mM KCl
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| - | 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
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| - | b. Cells were cultured on LB agar plate overnight at 370C.
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| - | c. 10-12 colonies were cultured in 250ml SOB medium.
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| - | d. It was incubated at 370C for 1hour. Then the flasks were transferred to 190C. It was incubated toll OD600 reached 0.5
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| - | e. Flask was placed in ice for 10min.
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| - | f. The cells were pelleted down by spinning at 4000rpm for 10min at 40C.
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| - | g. Cells were resuspended in 80ml ice cold TB(Transformation Buffer) and stored on ice for 10min.
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| - | h. It was centrifuged again at 4000rpm for 10min at 40C.
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| - | i. Pellet was resuspended in 20ml of TB with 1.5ml DMSO.
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| - | j. Final volume was aliquoted into microcentrifuge tubes (100-500µl) and stored at -800C.
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| | LET ME KNOW IF U WANT TO DO IT IN WIKI CODE! (THIS IS IN HTML) | | LET ME KNOW IF U WANT TO DO IT IN WIKI CODE! (THIS IS IN HTML) |
Revision as of 05:47, 18 October 2009
Indian Institute of Technology,Madras
LET ME KNOW IF U WANT TO DO IT IN WIKI CODE! (THIS IS IN HTML)
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