Q5® Site-Directed Mutagenesis Kit Quick Protocol (E0554)
Assemble the following reagents in a thin-walled PCR tube.
25 μl RXNFINAL CONC.Q5 Hot Start High-Fidelity 2X Master Mix12.5 μl1X10 μM Forward Primer1.25 μl0.5 μM10 μM Reverse Primer1.25 μl0.5 μMTemplate DNA (1–25 ng/μl)1 μl1-25 ngNuclease-free water9.0 μl.  
Mix reagents completely.
Transfer to a thermocycler and perform the following cycling conditions:
Thermocycling Conditions for a Routine PCR:STEPTEMPTIMEInitial Denaturation98°C30 seconds25 Cycles98°C10 seconds50–72°C*10–30 seconds72°C20–30 seconds/kbFinal Extension72°C2 minutesHold4–10°C. 
Assemble the following reagents: 
VOLUMEFINAL CONC.PCR Product1 μl 2X KLD Reaction Buffer5 μl1X10X KLD Enzyme Mix1 μl1XNuclease-free Water3 μl. 
Mix well by pipetting up and down.
Incubate at room temperature for 5 minutes.
Add 5 μl of the KLD mix from the "KLD Section" above to 50 μl of chemically-competent 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 40–100 μl onto appropriate selection plate. 
Incubate overnight at 37°C
