Team:Ionis Paris/Protocols 10

Protocol 11: Site directed Mutagenesis

Aim: Modify a given section of a plasmid

Exponential Amplification

Prepare the following reaction mix:

Perform a thermocycling as follows:

  • Initial denaturation: 98°C for 30s

  • 25 cycles of :
    - 98°C 10s
    - Provided Ta temperature 10-30s
    - 72°C; 20/30S per kB (amplification)

  • 72°C for 2 min

  • Hold: 4°C

  • Qsp 30 µL RNAse-free H2O

    • KLD Reaction

    Prepare the following reaction mix:

    Incubate for 5 min at room temperature

    Transformation

    Add 5 μl of KLD mix to 50 μl of chemically-competent cells.
    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.