Difference between revisions of "Team:Ionis Paris/Protocol 10"
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Revision as of 20:47, 16 October 2016
Prepare the following reaction mix: Perform a thermocycling as follows: Initial denaturation: 98°C for 30s 25 cycles of : 72°C for 2 min Hold: 4°C Qsp 30 µL RNAse-free H2O Prepare the following reaction mix: Incubate for 5 min at room temperature Add 5 μl of KLD mix to 50 μl of chemically-competent cells.
Protocol 10: Site directed Mutagenesis
Aim: Modify a given section of a plasmid
Exponential Amplification
- 98°C 10s
- Provided Ta temperature 10-30s
- 72°C; 20/30S per kB (amplification) KLD Reaction
Transformation
Incubate on ice for 30 minutes.
Heat shock at 42°C for 30 seconds.
Incubate on ice for 5 minutes.
Add 950 μl SOC, gently shake at 37°C for 1 hour.
Spread 40–100 μl onto appropriate selection plate, incubate overnight at 37°C.
