Q5® Site-Directed Mutagenesis Kit (Without Competent Cells) Protocol (E0552)
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ProtocolStep I: Exponential Amplification (PCR)
1. Assemble the following reagents in a thin-walled PCR tube.
|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|
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|
|Final Extension||72°C||2 minutes|
* 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.
Note: We suggest ensuring there is a clean PCR product by visualizing 2–5 μl of the reaction on an agarose gel.
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