Welcome guest, Login | Register

Home

X
加载中

This protocol is for making nucleotide changes at specific loci in a large vector (>= 10 kb), and based on QuikChange II XL Site-Directed Mutagenesis Kit (stratagene).

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

[Bio101] Site-Directed Mutagenesis

Molecular Biology > DNA > Mutagenesis
Author: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
2/5/2011, 9934 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.29

[Abstract] This protocol is for making nucleotide changes at specific loci in a large vector (>= 10 kb), and based on QuikChange II XL Site-Directed Mutagenesis Kit (stratagene).

Materials and Reagents

  1. QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene)
  2. LB agar (Sigma)
  3. Antibiotics (Sigma)

Equipment

  1. Thermal cyclers (Bio-Rad)

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

      Reaction Type
      Volume to Plate

      pWhitescript mutagenesis control

      250 μl
      pUC18 transformation control
      5 μl (in 200 μl NZY + broth)
      Sample mutagensis
      250 μl on each of two plates (entire transformation reaction)

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


How to cite this protocol: Jing, L. (2011). Site-Directed Mutagenesis. Bio-protocol Bio101: e29. DOI: 10.21769/BioProtoc.29; Full Text



Reproducibility Feedback:

  • Add Photo
  • Add Video

Bio-protocol's major goal is to make reproducing an experiment an easier task. If you have used this protocol, it would be great if you could share your experience by leaving some comments, uploading images or even sharing some videos. Please login to post your feedback.

Q&A and Troubleshooting:

  • Add Photo
  • Add Video

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.


Login | Register