Q5® Site-Directed Mutagenesis Kit Quick Protocol (E0554)
New England Biolabs
Published: 2022-02-10 DOI: 10.17504/protocols.io.bddei23e
Abstract
This is the quick protocol for the Q5® Site-Directed Mutagenesis Kit (E0554).
Before start
Primers should be designed with 5´ ends annealing back-to-back. We recommend using the NEB online design software, NEBaseChanger™.
Steps
Exponential Amplification (PCR)
1.
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 |
2.
Mix reagents completely.
3.
* For mutagenic primers, please use the Ta provided by the online NEB primer design software, * For mutagenic primers, please use the Ta provided by the online NEB primer design software, NEBaseChanger™..
Transfer to a thermalcycler and perform the following cycling conditions:
| 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 |
Note
Kinase, Ligase & DpnI (KLD) Treatment
4.
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 |
5.
Mix well by pipetting up and down.
6.
Incubate at Room temperature for 0h 5m 0s.
Transformation
7.
Add 5µL from previous step to 50µL.
8.
Incubate On ice for 0h 30m 0s.
9.
Heat shock at 42°C for 0h 0m 30s.
10.
Incubate 42On ice for 0h 5m 0s.
11.
Add 950µL.
12.
Gently shake at 37°C for 1h 0m 0s.
13.
Spread 40µL–100µL onto appropriate selection plate.
14.
Incubate 1h 0m 0s at 37°C.
