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From 2012.igem.org

Contents 1 Mutagenesis 1.1 Mutagenic Primer Design 1.2 Mutant Strand Synthesis Reaction (Thermal Cycling) 1.3 Dpn I Digestion of the Amplification Products 1.4 Transformation of XL10-Gold® Ultracompetent Cells

Mutagenesis

Using the QuikChange® Lightning Site-Directed Mutagenesis Kit

Mutagenic Primer Design

Synthesize two complimentary oligonucleotides containing the desired mutation, flanked by unmodified nucleotide sequence. Purify these oligonucleotide primers prior to use in the following steps.

Mutant Strand Synthesis Reaction (Thermal Cycling)

Prepare each sample reaction 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 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.

Transformation of XL10-Gold® Ultracompetent Cells

1. Gently thaw the XL10-Gold ultracompetent cells on ice. For each control and sample reaction to be transformed, aliquot 45 μl of the ultracompetent cells to a prechilled 14-ml BD Falcon polypropylene round-bottom tube.
2. Swirl the contents of the tube gently. Incubate the cells on ice for 2 minutes.
3. Transfer 2 μl of the Dpn I-treated DNA from each control and sample reaction to separate aliquots of the ultracompetent cells.
4. Swirl the transformation reactions gently to mix and incubate the reactions on ice for 30 minutes.
5. Heat-pulse the tubes in a 42°C water bath for 30 seconds. The duration of the heat pulse is critical for obtaining the highest efficiencies. Do not exceed 42°C.
6. Incubate the tubes on ice for 2 minutes.
7. Add 0.5 ml of preheated (42°C) SOC broth to each tube, then incubate the tubes at 37°C for 1 hour with shaking at 225–250 rpm.
8. Plate the appropriate volume of each transformation reaction, as indicated in the table below, on agar plates containing the appropriate antibiotic for the plasmid vector.