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

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Step I: Exponential Amplification (PCR)

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

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

3. Perform the following cycling conditions:

Thermocycling Conditions for a Routine PCR:
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.

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

1. Assemble the following reagents:
PCR Product 1 μl  
2X KLD Reaction Buffer 5 μl 1X
10X KLD Enzyme Mix 1 μl 1X
Nuclease-free Water 3 μl  

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

Step III: Transformation

1. Thaw a 50 μl aliquot of chemically competent E. coli cells [NEB 5-alpha Competent E. coli (High Efficiency), NEB #C2987, are recommended] 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