Team:Wageningen UR/Protocol/Mutagenesis

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== Dpn I Digestion of the Amplification Products ==
== Dpn I Digestion of the Amplification Products ==

Revision as of 11:15, 26 October 2012

Contents

Mutagenesis

QuikChange® Lightning Site-Directed Mutagenesis Kit

Prepare the sample reaction(s) as indicated below

  1. 5 μl of 10× reaction buffer
  2. X μl (10–100 ng) of dsDNA template
  3. X μl (125 ng) of oligonucleotide primer #1
  4. X μl (125 ng) of oligonucleotide primer #2
  5. 1 μl of dNTP mix
  6. 1.5 μl of QuikSolution reagent
  7. ddH2O to a final volume of 50 μl
  8. Then add: 1 μl of QuikChange® Lightning Enzyme

Cycling Parameters for the QuikChange Lightning Site-Directed Mutagenesis Method

    * For example, a 5-kb plasmid requires 2.5 minutes per cycle at 68°C.















Dpn I Digestion of the Amplification Products

  1. Add 2 μl of the provided Dpn I restriction enzyme directly to each amplification reaction.
  2. Gently and thoroughly mix each reaction mixture by pipetting the solution up and down several times. Briefly spin down the reaction mixtures and then immediately incubate at 37°C for 5 minutes to digest the parental (i.e., the nonmutated) supercoiled dsDNA.
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