Q5® Site-Directed Mutagenesis Kit (Without Competent Cells) Quick Protocol (E0552)

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

Protocol

Step 1: Exponential Amplification
  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  

Cycling Conditions:
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 mutagenic primers, please use the Ta provided by the online NEB primer design software, NEBaseChanger™.

Step 2: KLD Reaction
  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  
Incubate for 5 minutes at room temperature.

Step 3: Transformation
  1. Add 5 μl of KLD mix to 50 μl of chemically-competent E. coli cells.
  2. Incubate on ice for 30 minutes.
  3. Heat shock at 42°C for 30 seconds.
  4. Incubate on ice for 5 minutes.
  5. Add 950 μl SOC, gently shake at 37°C for 1 hour.
  6. Spread 40–100 μl onto appropriate selection plate, incubate overnight at 37°C.