Feb 10, 2022
Version 2
Q5® Site-Directed Mutagenesis Kit Quick Protocol (E0554) V.2
- 1New England Biolabs
- New England Biolabs (NEB)Tech. support phone: +1(800)632-7799 email: info@neb.com
External link: https://www.neb.com/protocols/2013/01/26/q5-site-directed-mutagenesis-kit-quick-protocol-e0554
Protocol Citation: New England Biolabs 2022. Q5® Site-Directed Mutagenesis Kit Quick Protocol (E0554). protocols.io https://dx.doi.org/10.17504/protocols.io.bddei23eVersion created by New England Biolabs
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: March 08, 2020
Last Modified: February 10, 2022
Protocol Integer ID: 33926
Keywords: site directed mutagenesis, exponential amplification for SDM, E0554, SDM
Abstract
This is the quick protocol for the Q5® Site-Directed Mutagenesis Kit (E0554).
Materials
MATERIALS
Q5 Site-Directed Mutagenesis Kit - 10 rxnsNew England BiolabsCatalog #E0554S
Safety warnings
Please refer to Safety Data Sheets (SDS) for health and environmental hazards.
Before start
Primers should be designed with 5´ ends annealing back-to-back. We recommend using the NEB online design software, NEBaseChanger™.
Exponential Amplification (PCR)
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 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
* For mutagenic primers, please use the Ta provided by the online NEB primer design software, NEBaseChanger™.
Kinase, Ligase & DpnI (KLD) Treatment
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 00:05:00 .
Transformation
Transformation
Add 5 µL KLD mix from previous step to 50 µL chemically-competent cells .
Incubate On ice for 00:30:00 .
Heat shock at 42 °C for 00:00:30 .
Incubate On ice for 00:05:00 .
Add 950 µL SOC .
Gently shake at 37 °C for 01:00:00 .
Spread 40 µL –100 µL onto appropriate selection plate.
Incubate Overnight at 37 °C .