Assemble the following reagents in a thin-walled PCR tube.

A	B	C
	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:

A	B	C
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

• For a Q5-optimized annealing temperature of mutagenic primers, please use NEBaseChanger™, the online NEB primer design software. For pre-designed, back-to-back primer sets, a Ta = Tm + 3 rule can be applied, but optimization may be necessary.

Assemble the following reagents:

A	B	C
	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 0h 5m 0s.

Thaw a tube of NEB 5-alpha Competent E. coli cells On ice.

Add 5µL 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 0h 30m 0s.

Heat shock at 42°C for 0h 0m 30s.

Place 42On ice for 0h 5m 0s.

Pipette 950µL into the mixture.

Incubate at 37°C for 1h 0m 0s with shaking (250rpm,0h 0m 0s).

Mix the cells thoroughly by flicking the tube and inverting.

Spread 50µL-100µL onto a selection plate.

Incubate 1h 0m 0s at 37°C.