Q5® Site-Directed Mutagenesis (E0552)
Assemble the following reagents in a thin-walled PCR tube.
Mix reagents completely.
Transfer to a thermocycler and perform the following cycling conditions:
Assemble the following reagents: Mix well by pipetting up and down.
Incubate at room temperature for 5 minutes.
Thaw a 50 μl aliquot of chemically competent E. coli cells on ice.
Add 5 μl of the KLD mix from the "KLD Section" above to the tube of thawed cells.
Carefully flick the tube 4-5 times to mix.
Do not vortex.
Place the mixture on ice for 30 minutes.
Heat shock at 42°C for 30 seconds.
Place on ice for 5 minutes.
Pipette 950 μl of room temperature SOC into the mixture.
Incubate at 37°C for 60 minutes with shaking (250 rpm).
Mix the cells thoroughly by flicking the tube and inverting.
Spread 50-100 μl onto a selection plate.
Incubate overnight at 37°C
