Difference between revisions of "Team:IISc-Bangalore/Protocols"
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| − | <td>From this point, cells should always be placed in the cold (below 4°C), all buffers should be ice-cold, and all plasticware/glassware should be pre-chilled.</td> | + | <td colspan="3">From this point, cells should always be placed in the cold (below 4°C), all buffers should be ice-cold, and all plasticware/glassware should be pre-chilled.</td> |
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Revision as of 12:28, 22 October 2017
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Contents
Transformation
Preparation of competent cells - TSS method
Day 1
| Step | Description | Rationale |
|---|---|---|
| 1 | Prepare a primary inoculum of E. coli in 5 mL LB in a test tube. | - |
| 2 | Incubate overnight at 37°C, 170 rpm. | - |
Day 2
| Step | Description | Comments |
|---|---|---|
| 1 | Inoculate 1 mL overnight culture in 100 mL LB medium (1% inoculum) | Depending on how many aliquots of competent cells you wish to make, you can vary the volume of LB medium used. This protocol yields 1 aliquot per mL of LB medium. |
| 2 | Incubate at 37°C, 170 rpm until the OD reaches 0.4 (~1 h 45 min for E. coli DH5α cells) | This is the early exponential phase; cells are physiologically ideal for the preparation of competent cells. |
| From this point, cells should always be placed in the cold (below 4°C), all buffers should be ice-cold, and all plasticware/glassware should be pre-chilled. | ||
| 3 | Place the culture at 4°C for 45 min | - |
| 4 | Spin down the culture at 10000 rpm, 10 min, 4°C | - |
| 5 | Resuspend the cell pellet in 1 mL ice-cold TSS buffer | - |
| 6 | Spin down the culture at 10000 rpm, 10 min, 4°C | - |
| 7 | Make 50-100 uL aliquots in chilled microfuge tubes, snap-freeze in liquid nitrogen and store at -80°C for long-term storage | - |
