Team:Wageningen UR/Protocol/Mutagenesis
From 2012.igem.org
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mixtures and then immediately incubate at 37°C for 5 minutes to digest | mixtures and then immediately incubate at 37°C for 5 minutes to digest | ||
the parental (i.e., the nonmutated) supercoiled dsDNA.</li> | the parental (i.e., the nonmutated) supercoiled dsDNA.</li> | ||
+ | </ol> | ||
+ | <br/> | ||
+ | |||
+ | == Transformation of XL10-Gold® Ultracompetent Cells == | ||
+ | <ol> | ||
+ | <li>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.</li> | ||
+ | <li>Add 2 μl of the β-ME mix provided with the kit to the 45 μl of | ||
+ | cells.</li> | ||
+ | <li>Swirl the contents of the tube gently. Incubate the cells on ice for | ||
+ | 2 minutes.</li> | ||
+ | <li>Transfer 2 μl of the Dpn I-treated DNA from each control and sample | ||
+ | reaction to separate aliquots of the ultracompetent cells.</li> | ||
+ | <li>Swirl the transformation reactions gently to mix and incubate the | ||
+ | reactions on ice for 30 minutes.</li> | ||
+ | <li>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.</li> | ||
+ | <li>Incubate the tubes on ice for 2 minutes.</li> | ||
+ | <li>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.</li> | ||
+ | <li>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.</li> | ||
</ol> | </ol> |
Revision as of 11:22, 26 October 2012
Contents |
Mutagenesis
QuikChange® Lightning Site-Directed Mutagenesis Kit
Prepare the sample reaction(s) as indicated below
- 5 μl of 10× reaction buffer
- X μl (10–100 ng) of dsDNA template
- X μl (125 ng) of oligonucleotide primer #1
- X μl (125 ng) of oligonucleotide primer #2
- 1 μl of dNTP mix
- 1.5 μl of QuikSolution reagent
- ddH2O to a final volume of 50 μl
- Then add: 1 μl of QuikChange® Lightning Enzyme
Cycling Parameters for the QuikChange Lightning Site-Directed Mutagenesis Method
Dpn I Digestion of the Amplification Products
- Add 2 μl of the provided Dpn I restriction enzyme directly to each amplification reaction.
- 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
- 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.
- Add 2 μl of the β-ME mix provided with the kit to the 45 μl of cells.
- Swirl the contents of the tube gently. Incubate the cells on ice for 2 minutes.
- Transfer 2 μl of the Dpn I-treated DNA from each control and sample reaction to separate aliquots of the ultracompetent cells.
- Swirl the transformation reactions gently to mix and incubate the reactions on ice for 30 minutes.
- 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.
- Incubate the tubes on ice for 2 minutes.
- 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.
- 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.