Oct 13, 2024

Public workspaceSite-Directed Mutagenesis

DOI
dx.doi.org/10.17504/protocols.io.81wgbzxjygpk/v1
  • 1Washington University
Cecilia Escudero
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DOI: dx.doi.org/10.17504/protocols.io.81wgbzxjygpk/v1
Protocol Citation: Carolina Lopez 2024. Site-Directed Mutagenesis. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbzxjygpk/v1
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: May 28, 2024
Last Modified: October 13, 2024
Protocol Integer ID: 100757
Abstract
This protocols describes the procedure for site-directed mutagenesis by PCR
Materials
Reagents:
  • Q5 SDM Kit (NEB, E0554S)
  • Competent E. coli cells (NEB, C3040H)
  • LB plates with necessary antibiotic
SDM PCR Protocol
SDM PCR Protocol
1h 39m
1h 39m
1. Mix in Amount0.2 mL PCR Tube:

2. Run PCR program:


Kinase, Ligase, and Dpn1 (KLD) Reaction:
3. Mix in Amount0.2 mL PCR Tube:

4. Mix well by pipetting up and down and incubate for Duration00:15:00 at room temperature.
5. Transform Product into E. coli
a) Add Amount2 µL of product to Amount50 µL of TOP10 cells and pipette up and down slowly to mix.
b) Incubate for Duration00:20:00 on ice.
c) Heat shock bacteria in Temperature42 °C waterbath for Duration00:01:00 .
d) Incubate on Ice for Duration00:03:00 .
e) Add Amount100 µL of SOC media and shake in warm room for Duration01:00:00 .
f) Plate bacteria onto LB-Antibiotic Plate and spread cells with plate spreader to get individual colonies.
g) Incubate overnight in Temperature37 °C warm room.
h) Pick colonies for miniprep growth and sequencing.
1h 39m
Protocol references
Site-Directed Mutagenesis:
Q5‱ Site-Directed Mutagenesis Kit | NEB

Primer design:
  • Helpful tool for designing primers is https://nebasechanger.neb.com/
  • Just insert vector sequence and indicate residues that need to be deleted, inserted, or mutated.