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Site-directed Mutagenesis Using Dpn1

LJ Lili Jing

Published: Feb 5, 2011 DOI: 10.21769/BioProtoc.29 Views: 22776

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Abstract

Site-directed mutagenesis is an important and widely used tool in molecular biology to generate specific changes in the DNA sequence of a given gene/genome. The protocol described here is for making nucleotide changes at specific loci in a large vector (>= 10 kb), and is based on the QuikChange II XL Site-Directed Mutagenesis Kit.

Materials and Reagents

  1. Dpn1 restriction enzyme
  2. XL10-Gold Ultracompetent Cells
  3. QuikChange II XL Site-Directed Mutagenesis Kit (Guidechem)
  4. LB agar (Sigma-Aldrich)
  5. Antibiotics (Sigma-Aldrich)
  6. NZ amine (casein hydrolysate)
  7. Yeast extract
  8. NaCl
  9. NaOH
  10. MgCl2
  11. MgSO4
  12. Glucose
  13. NZY+ broth (see Recipes)

Equipment

  1. Thermal cyclers (Bio-Rad Laboratories)
  2. Bench-top centrifuge

Procedure

Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.

Category

Molecular Biology > DNA > Mutagenesis

Molecular Biology > DNA > Genotyping

Molecular Biology > DNA > PCR

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I checked this protocol months ago, but I didn’t use this method,...
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