Q5® Site - Directed Mutagenesis Kit Quick Protocol ( E0554 )
Assemble the following reagents in a thin - walled PCR tube .
25 μl RXN ; FINAL CONC . ; Q5 Hot Start High - Fidelity 2X Master Mix 12.5 μl 1X ; 10 μM Forward Primer 1.25 μl 0.5 μM ; 10 μM Reverse Primer 1.25 μl 0.5 μM ; Template DNA ( 1–25 ng / μl ) 1 μl 1 - 25 ng ; Nuclease - free water 9.0 μl .
Mix reagents completely .
Transfer to a thermocycler and perform the following cycling conditions :
Thermocycling Conditions for a Routine PCR : STEP TEMP TIME Initial Denaturation 98°C 30 seconds 25 Cycles 98°C 10 seconds 50–72°C * 10–30 seconds 72°C 20–30 seconds / kb Final Extension 72°C 2 minutes Hold 4–10°C .
Assemble the following reagents :
VOLUME FINAL CONC . PCR Product 1 μl ; 2X KLD Reaction Buffer 5 μl 1X ; 10X KLD Enzyme Mix 1 μl 1X ; Nuclease - free Water 3 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
