McClean: Q5 Mutagenesis Kit from NEB

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The protocol for NEB's Q5 mutagenesis kit is listed below and also comes with the kit. This page is for users to add tips and tricks to increase mutation efficiency. Protocol:Protocol

Step I: Exponential Amplification (PCR)

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

Reagent Volume
Q5 Hot Start High-Fidelity 2X Master Mix 12.5μL
10 μM Forward Primer 1.25μL
10 μM Reverse Primer 1.25μL
Template DNA (1–25 ng/μl) 1μL
Sterile Water 9μL
Total Volume 25μL

2. Mix reagents completely, then transfer to a thermocycler.

3. Perform the following cycling conditions:

Thermocycling Conditions for a Routine PCR:

  • 98°C 30 seconds
  • 25 Cycles of the following
    • 98°C 10 seconds
    • 50–72°C* 10–30 seconds
    • 72°C 20–30 seconds/kb
  • 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.

Step II: Kinase, Ligase & DpnI (KLD) Treatment

1. Assemble the following reagents:

Reagent Volume
PCR Product 1μL
2X KLD Reaction Buffer 5μL
10X KLD Enzyme Mix 1μL
Sterile Water 3μL
Total Volume 10μL

2. Mix well by pipetting up and down and incubate at room temperature for 5 minutes.

Step III: Transformation

  • 1. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice.
  • 2. Add 5 μl of the KLD mix from Step II to the tube of thawed cells. Carefully flick the tube 4-5 times to mix. Do not vortex.
  • 3. Place the mixture on ice for 30 minutes.
  • 4. Heat shock at 42°C for 30 seconds.
  • 5. Place on ice for 5 minutes.
  • 6. Pipette 950 μl of room temperature SOC into the mixture.
  • 7. Incubate at 37°C for 60 minutes with shaking (250 rpm).
  • 8. Mix the cells thoroughly by flicking the tube and inverting, then spread 50-100 μl onto a selection plate and incubate overnight at 37°C. It may be necessary (particularly for simple substitution and deletion experiments) to make a 10- to 40-fold dilution of the transformation mix in SOC prior to plating, to avoid a lawn of colonies


  • SG 4/24/2017- When my plasmids were not mutating I ordered and used a new kit and that fixed the problem. I think that at some point that DPNI that is in the KLD buffer loses it's affect because I get more colonies and many of them are not mutated compared to when I use a new kit.
  • SG 4/24/2017- I would recommend checking the PCR on a gel before continuing with the ligation to make sure the PCR worked. It doesn't say to do that but it has saved me time and resources by checking.