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
