Exponential amplification

Primers should be designed with 5´ ends annealing back-to-back. It is recommend to use the NEB online design software, NEBaseChanger™.

Prepare the following reaction mix:

Component	Volume	Final concentration
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 µL

Perform a thermocycling as follows:

Initial denaturation: 98°C for 30s

25 cycles of :
- 98°C 10s
- Provided Ta temperature 10-30s
- 72°C; 20/30s per kB (amplification)

72°C for 2 min

Hold: 4°C

KLD Reaction

Prepare the following reaction mix:

Component	Volume	Final concentration
PCR product	1 µL
2X KLD Reaction Buffer	5 µL	1X
10X KLD Enzyme Mix	1 µL	1X
Nuclease-free Water	3 µL

Incubate for 5 min at room temperature

Transformation

Add 5μL of KLD mix to 50μL of chemically-competent cells.
Incubate on ice for 30 minutes.
Heat shock at 42°C for 30 seconds.
Incubate on ice for 5 minutes.
Add 950μL SOC, gently shake at 37°C for 1 hour.
Spread 40–100 μL onto appropriate selection plate, incubate overnight at 37°C.