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Site-directed Mutagenesis Using Dpn1   

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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

  1. Mutant Strand Synthesis Reaction
    1. Prepare the sample reaction as follows:
      5 μl of 10x reaction buffer
      X μl (10 ng) of dsDNA template
      1.25 μl (125 ng) of oligonucleotide primer #1
      1.25 μl (125 ng) of oligonucleotide primer #2
      1 μl of dNTP mix
      3 μl of QuikSolution
      ddH2O to a final volume of 50 μl
      Then add 1 μl of PfuUltra HF DNA polymerase (2.5 U/μl)
    2. Cycle each reaction using the following parameters (be sure to adhere to the 18 cycle limit)

      Segment
      Cycles
      Temperature
      Time
      1
      1
      95 °C
      1 min
      2
      18
      95 °C
      50 sec
      60 °C
      50 sec
      68 °C
      1 min/kb of plasmid length*
      3
      1
      68 °C
      7 min

      *for example, a 5 kb plasmid requires 5 min at 68 °C per cycle
    3. Following cycling, place reaction tubes on ice for 2 min to cool the reactions to 37 °C
    4. Check product by electrophoresis of 10 μl of product on 1% agarose gel. While gel is running, start Dpn1 Digestion

  2. Dpn1 Digestion of the Amplification Products
    1. Add 1 μl of the Dpn1 restriction enzyme (10 U/μl) directly to each amplification reaction using a small, pointed pipet tip
    2. Gently mix each reaction mixture by pipetting up and down several times. Spin for 1 min and incubate the reaction at 37 °C for 1 h to digest the parental supercoiled dsDNA

  3. Transformation of XL10-Gold Ultracompetent Cells
    1. Gently thaw XL10-Gold ultracompetent cells on ice. For each control and sample reaction to be transformed, aliquot 45 μl of the cells to a prechilled polypropylene tube
    2. Add 2 μl of the B-ME mix provided with the kit to the 45 μl cells
    3. Swirl the contents of the tube gently. Incubate on ice for 10 min, swirling gently every 2 min
    4. Transfer 2 μl of the Dpn1-treated DNA from each control and sample reaction to separate aliquots of the ultracompetent cells
      For a positive control, add 1 μl of 0.01 ng/μl pUC18 control plasmid
    5. Preheat NZY + broth in a 42 °C water bath
    6. Heat-pulse tubes in a 42 °C water bath for 30 sec
    7. Incubate the tubes on ice for 2 min
    8. Add 0.5 ml of preheated (42 °C) NZY + broth to each tube, then incubate the tubes at 37 °C for 1 h with shaking 225-250 rpm
    9. Plate the appropriate volume of each transformation rxn as indicated on the table below on proper selection plates


    10. Incubate plates at 37 °C for > 16 h

Recipes

  1. NZY+ Broth (per Liter)
    10 g of NZ amine (casein hydrolysate)
    5 g of yeast extract
    5 g of NaCl
    Add deionized H2O to a final volume of 1 L
    Adjust to pH 7.5 using NaOH and then autoclave
    Add the following filer-sterilized supplements prior to use:
    12.5 ml of 1 M MgCl2
    12.5 ml of 1 M MgSO4
    20 ml of 20% (w/v) glucose (or 10 ml of 2 M glucose)

Acknowledgments

This protocol was developed in the Michael Granato Lab at University of Pennsylvania, Philadelphia, USA, and modified from the manual of QuikChange II XL Site-Directed Mutagenesis Kit. This work was supported by NIH grant R01HD037975.

Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Jing, L. (2011). Site-directed Mutagenesis Using Dpn1. Bio-protocol Bio101: e29. DOI: 10.21769/BioProtoc.29.
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