Protocol for Q5® Site-Directed Mutagenesis Kit (E0554)
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Protocol
Step 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:
| 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 |
Step II: Kinase, Ligase & DpnI (KLD) Treatment
1. Assemble the following reagents:
| 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 |
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
