From 2009.igem.org
(Difference between revisions)
|
|
Line 34: |
Line 34: |
| <div id="aContent"> | | <div id="aContent"> |
| | | |
- | PROTOCOLS
| |
- |
| |
- | 1) ULTRACOMPETENT CELL PREPARATION
| |
- | a. Materials/Buffers
| |
- | i. 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
| |
- | ii. TRANSFORMATION BUFFER (TB) 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
| |
- | b. Cells were cultured on LB agar plate overnight at 370C.
| |
- | c. 10-12 colonies were cultured in 250ml SOB medium.
| |
- | d. It was incubated at 370C for 1hour. Then the flasks were transferred to 190C. It was incubated toll OD600 reached 0.5
| |
- | e. Flask was placed in ice for 10min.
| |
- | f. The cells were pelleted down by spinning at 4000rpm for 10min at 40C.
| |
- | g. Cells were resuspended in 80ml ice cold TB(Transformation Buffer) and stored on ice for 10min.
| |
- | h. It was centrifuged again at 4000rpm for 10min at 40C.
| |
- | i. Pellet was resuspended in 20ml of TB with 1.5ml DMSO.
| |
- | j. Final volume was aliquoted into microcentrifuge tubes (100-500µl) and stored at -800C.
| |
| | | |
| 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)