Q5® Site-Directed Mutagenesis (E0554)
New England Biolabs
Abstract
This is the protocol for the Q5® Site-Directed Mutagenesis Kit (E0554).
Before start
Steps
Exponential Amplification (PCR)
Assemble the following reagents in a thin-walled PCR tube.
| A | B | C |
|---|---|---|
| 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 |
Mix reagents completely.
Transfer to a thermocycler and perform the following cycling conditions:
Thermocycling Conditions for a Routine PCR:
| A | B | C |
|---|---|---|
| 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 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.
Kinase, Ligase & DpnI (KLD) Treatment
Assemble the following reagents:
| A | B | C |
|---|---|---|
| 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 |
Mix well by pipetting up and down.
Incubate at Room temperature for 0h 5m 0s.
Transformation
Thaw a tube of NEB 5-alpha Competent E. coli cells On ice.
Add 5µL from the "KLD Section" above to the tube of thawed cells.
Carefully flick the tube 4-5 times to mix. Do not vortex.
Place the mixture On ice for 0h 30m 0s.
Heat shock at 42°C for 0h 0m 30s.
Place 42On ice for 0h 5m 0s.
Pipette 950µL into the mixture.
Incubate at 37°C for 1h 0m 0s with shaking (250rpm,0h 0m 0s).
Mix the cells thoroughly by flicking the tube and inverting.
Spread 50µL-100µL onto a selection plate.
Incubate 1h 0m 0s at 37°C.
