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.
