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From 2011.igem.org
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4. Incubate on ice for 30 min. | 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'') | + | 5. Heat shock the cells at 42°C for exactly 60 seconds and immediately return them to ice.(''this heat shock time is optimized for E.coli K-12, JM109 strands'') |
| - | 6. Incubate 5 minutes in ice | + | 6. Incubate 5 minutes in ice. |
7. Add 200ul of room temperature SOC medium to the microtubes. (''It is no longer necessary to keep the cells on ice from this step onwards'') | 7. Add 200ul of room temperature SOC medium to the microtubes. (''It is no longer necessary to keep the cells on ice from this step onwards'') | ||
Latest revision as of 00:45, 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 at 42°C for exactly 60 seconds and immediately return them to ice.(this heat shock time is optimized for E.coli K-12, JM109 strands)
6. Incubate 5 minutes in ice.
7. Add 200ul of room temperature SOC medium to the microtubes. (It is no longer necessary to keep the cells on ice from this step onwards)
8. Incubate for 1-2 hours at 37°C with vigorous shaking.
9. Spread 100ul of the transformants onto an agar plate with the appropriate antibiotic.
10. Incubate at 37°C "overnight" with the top side looking downwards.
