Q5® Site-Directed Mutagenesis (E0552)
New England Biolabs
Abstract
The Q5 Site-Directed Mutagenesis Kit (Without Competent Cells) enables rapid, site-specific mutagenesis of double-stranded plasmid DNA in less than 2 hours (Figure 1). The kit utilizes the robust Q5 Hot Start High-Fidelity DNA Polymerase along with custom mutagenic primers to create insertions, deletions and substitutions in a wide variety of plasmids. After PCR, the amplified material is added directly to a unique Kinase-Ligase-DpnI (KLD) enzyme mix for rapid (5 minutes), room temperature circularization and template removal (Figure 2). Transformation into high-efficiency chemically-competent E. coli , not supplied, ensures robust results with plasmids up to at least 20 kb in length. Kit is available with competent cells (NEB #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 |
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 50µL 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 0h 5m 0s at 37°C.
