Feb 10, 2022

Public workspaceQ5® Site-Directed Mutagenesis Kit Quick Protocol (E0554) V.2

  • 1New England Biolabs
  • New England Biolabs (NEB)
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Protocol CitationNew 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 07, 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
ReagentQ5 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.
ABC
25 μl RXNFINAL CONC.
Q5 Hot Start High-Fidelity 2X Master Mix12.5 μl1X
10 μM Forward Primer1.25 μl0.5 μM
10 μM Reverse Primer1.25 μl0.5 μM
Template DNA (1–25 ng/μl)1 μl1-25 ng
Nuclease-free water9.0 μl

Pipetting
Mix reagents completely.
Mix
Transfer to a thermalcycler and perform the following cycling conditions:
ABC
STEPTEMPTIME
Initial Denaturation98°C30 seconds
25 Cycles98°C10 seconds
50–72°C*10–30 seconds
72°C20–30 seconds/kb
Final Extension72°C2 minutes
Hold4–10°C

Note
* For mutagenic primers, please use the Ta provided by the online NEB primer design software, NEBaseChanger™.
PCR
Kinase, Ligase & DpnI (KLD) Treatment
Kinase, Ligase & DpnI (KLD) Treatment
Assemble the following reagents:
ABC
VOLUMEFINAL CONC.
PCR Product1 μl
2X KLD Reaction Buffer5 μl1X
10X KLD Enzyme Mix1 μl1X
Nuclease-free Water3 μl

Pipetting
Mix well by pipetting up and down.
Mix
Incubate at TemperatureRoom temperature for Duration00:05:00 .
Incubation
Transformation
Transformation
Add Amount5 µL KLD mix from previous step to Amount50 µL chemically-competent cells .
Pipetting
Incubate TemperatureOn ice for Duration00:30:00 .
Incubation
Heat shock at Temperature42 °C for Duration00:00:30 .
Incubate TemperatureOn ice for Duration00:05:00 .
Incubation
Add Amount950 µL SOC .
Pipetting
Gently shake at Temperature37 °C for Duration01:00:00 .
Spread Amount40 µL Amount100 µL onto appropriate selection plate.
Pipetting
Incubate DurationOvernight at Temperature37 °C .
Incubation