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Transformation
From 2011.igem.org
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| - | + | Materials: | |
| + | |||
| + | Chemically competent cells | ||
| + | |||
| + | DNA plasmid ligation | ||
| + | |||
| + | SOC medium | ||
| + | |||
| + | LB+Agar plater with the appropriate antibiotic | ||
| + | |||
| + | Procedure: | ||
| + | |||
| + | 1. Thaw the competent cells on ice for 5min. ( competent cells are extremely susceptible to heat, work with them ALWAYS on ice unless specified not to) | ||
| + | |||
| + | 2. Meanwhile the cells are thawing out, add 2-4ul of DNA to a 2ml microtube on ice. (use molecular grade H2O for your negative control) | ||
| + | |||
| + | 3. Add 50ul of the thawed out competent cells to to 2ml microtube | ||
| + | |||
| + | 4. Incubate on ice for 30 min. | ||
| + | |||
| + | 5. Heat shock the cells for exactly 60 seconds and immediately return them to ice.(this heat shock time is optimized for E.coli K-12, JM109 strands) | ||
[https://2011.igem.org/Team:Panama/Protocols/Wetlab '''Back'''] | [https://2011.igem.org/Team:Panama/Protocols/Wetlab '''Back'''] | ||
Revision as of 00:29, 29 September 2011
Materials:
Chemically competent cells
DNA plasmid ligation
SOC medium
LB+Agar plater with the appropriate antibiotic
Procedure:
1. Thaw the competent cells on ice for 5min. ( competent cells are extremely susceptible to heat, work with them ALWAYS on ice unless specified not to)
2. Meanwhile the cells are thawing out, add 2-4ul of DNA to a 2ml microtube on ice. (use molecular grade H2O for your negative control)
3. Add 50ul of the thawed out competent cells to to 2ml microtube
4. Incubate on ice for 30 min.
5. Heat shock the cells for exactly 60 seconds and immediately return them to ice.(this heat shock time is optimized for E.coli K-12, JM109 strands)
