Editing Quick Change (section)

= New Protocol: Lightning Quick Change  =



Mutant Strand Synthesis Reaction (Thermal Cycling)<br>Notes: Ensure that the plasmid DNA template is isolated from a dam+ E. coli strain. The majority of the commonly used E. coli strains are dam+. Plasmid DNA isolated from damâ strains (e.g. JM110 and SCS110) is not suitable.



To maximize temperature-cycling performance, we strongly recommend using thin-walled tubes, which ensure ideal contact with the temperature cyclerâs heat blocks. The following protocols were optimized using thin-walled tubes.

1. Synthesize two complimentary oligonucleotides containing the desired mutation, flanked by unmodified nucleotide sequence. Purify these oligonucleotide primers prior to use in the following steps (see Mutagenic Primer Design).

2. Prepare the control reaction as indicated below:

5 ÎŒl of 10Ã reaction buffer<br>5 ÎŒl (25 ng) of pWhitescript 4.5-kb control plasmid (5 ng/ÎŒl)<br>1.25 ÎŒl (125 ng) of oligonucleotide control primer #1<br>[34-mer (100 ng/ÎŒl)]<br>1.25 ÎŒl (125 ng) of oligonucleotide control primer #2<br>[34-mer (100 ng/ÎŒl)]<br>1 ÎŒl of dNTP mix<br>1.5 ÎŒl of QuikSolution reagent<br>34 ÎŒl ddH2O (to bring the final reaction volume to 50 ÎŒl)

Then add:<br>1 ÎŒl of QuikChange Lightning Enzyme

3.Prepare the sample reaction(s) as indicated below:<br>Note: Set up a series of sample reactions using various amounts of dsDNA template ranging from 10 to 100 ng (e.g., 10, 25, 50, and 100 ng of dsDNA template) while keeping the primer concentration constant.

5 ÎŒl of 10Ã reaction buffer<br>X ÎŒl (10â100 ng) of dsDNA template<br>X ÎŒl (125 ng) of oligonucleotide primer #1<br>X ÎŒl (125 ng) of oligonucleotide primer #2<br>1 ÎŒl of dNTP mix<br>1.5 ÎŒl of QuikSolution reagent<br>ddH2O to a final volume of 50 ÎŒl

Then add:<br>1 ÎŒl of QuikChange Lightning Enzyme

<br>1: 1 cycle of 95°C 2 minutes<br>2: 18 cycles 95°C 20 seconds, 60°C 10 seconds, 68°C 30 seconds/kb of plasmid length*<br>3: 1 cycle of 68°C for 5 minutes

Dpn I Digestion of the Amplification Products:

1. Add 2 ÎŒl of the provided Dpn I restriction enzyme directly to each amplification reaction.

Notes: Use only the Dpn I enzyme provided; do not substitute with an enzyme from another source.

If a mineral oil overlay was used during the synthesis reaction, it is important to insert the pipet tip below the overlay when adding the Dpn I enzyme to the reaction tubes and when transferring the 2 ÎŒl of Dpn I-treated DNA to the transformation reaction. Using specialized aerosol-resistant pipet tips, which are small and pointed, facilitates this process.<br>

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