QuikChangeII Mutagenesis Kit from Agilent Technologies

March 01, 2013

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Colonies of XL01-Blue on an LB agar plate supplemented with the appropriate antibiotic for selection of the plasmid (ampicillin). The results of transformations of two consecutive mutagenesis reactions at the C-terminus of a protein of interest are shown in Panel A. The first one (mut1) causes the substitution of adjacent Lys and Leu to Val and Thr residues respectively, while the second one (mut2) implies the substitution of Phe and Met residues to Ile and a stop codon respectively, causing in this way the C-terminal truncation of the protein. In this case, DNA sequencing has been used for screening; chromatograms of DNA sequences of positive clones for both reactions are reported in Panel B. DNA and protein sequence alignment of the wild-type and mutated sequences is reported in Panel C; only part of the sequences, containing the mutations of interest (KL-->VT in red, FM-->Istop in blue), is shown. Expression test on E.coli BL21 confirm the partial truncation of the protein for the mut2 mutant (see SDS-PAGE on Panel D; bands corresponding to the over-expressed wt and mut2 proteins are high-lighted with red boxes).

Company:

Agilent Technologies

Product Name:

QuikChangeII Site-Directed Mutagenesis Kit

Catalog Number:

200553

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This kit allows the introduction of desired mutations (point mutations, deletion or insertion of single or multiple adjacent nucleic acids) into DNA that has been inserted into a plasmid. The method is based on DNA amplification using a high-fidelity DNA polymerase and two synthetic oligonucleotide primers, each containing the desired mutation. Extension of the oligonucleotide primers generates a mutated plasmid containing staggered nicks. Primers can be easily designed using the software from Agilent’s website (registration is required and use is free of charge), by simply uploading the DNA sequence of the vector of interest and selecting the nucleotide/amino acid residue(s) to mutate. The resulting amplification product is than treated with DpnI endonuclease, which is specific for methylated and hemimethylated DNA. In this way, the parental DNA template is digested and it is possible to select for the mutation‐containing synthesized DNA, by transformation of the reaction into E. coli competent cells.

Experimental Design and Results Summary

Application

Site-directed mutagenesis

Starting Material

Plasmid DNA

Protocol Overview

Amplification is carried out using 50 uL reactions and following the manufacturer's instructions. For medium sized plasmid templates (4000-6000 bp) 50 ng of DNA are used as input, and 125 ng of each primer are used. Usually, increasing the DNA concentration is not recommended and could increase the number of false positives during the screening step. Primers are purchased separately and are not included in the kit (a standard scale synthesis of 25 ng or less is sufficient; the use of desalted or even HPLC-purified oligonucteotides is strongly recommended, the latter in the case of long primers). Typically, for routine mutagenesis (single nucleotide substitutions), the suggested amplification reaction set-up is used, with annealing temperature of 55°C for 16 cycles. For more extensive mutagenesis (multiple nucleic acid substitutions or large deletion/insertion) or when long mutagenesis primers are used (more than 40 bases), adjustments to the amplification protocol are necessary. In particular, the annealing temperature is increased to 60°C or even 65°C, depending on the length of the primers, and 18 amplification cycles are run. The time of annealing steps is always 1 min. The extension temperature is fixed (68°C), while extension time depends on the length of the plasmid DNA template to mutate (1min/1kb). Digestion with DpnI is usually carried out at 37°C for 1 h; prolonging digestion time could reduce the number of false-positives during the screening. The concentration of DpnI enzyme can be reduced without significant effect on the digestion rate (usually 0.5 uL for 1.5h instead of 1 uL for 1h). XL01-Blue Supercompetent cells are used for transformation of the digested DNA (25 uL of cells are sufficient for a single reaction. After addition of the DNA, cells are incubated on ice for 20-30min and then heat-shocked at 42°C for 45sec; after a brief incubation on ice for 2 min, 100 uL of SOC medium are added and cells are incubated at 37°C for 1h with shaking at 200 rpm. All the culture volume is plated on an LB agar Petri dish, containing proper antibiotic for the selection of the plasmid of interest). Avoiding freeze -thawing of the cells is essential; this could dramatically affect transformation efficiency and consequently, the mutation rate. For this reason, aliquoting XL01-Blue cells is highly recommended. Cells are grown for at least 14-16h; screening of the positives clones is usually carried out by DNA sequencing or, in the case of large deletions, by colony PCR amplification using specific primer pairs. In the case of addition/suppression of specific restriction sites, screening can be made by digestion with endonucleases.

Tips

Increasing the total extension time by 1 additional minute can increase the number of positive mutants. Also to increase the number of positive clones, ethanol precipitate DNA prior to transformation (for a reaction volume of 50 uL, 100 uL of EtOH and 5uL of 3M sodium acetate are added; solution is mixed, incubated at -80°C for 30min at centrifuged at 13.000 rpm for 20 min. Supernatant is discarded and DNA pellet is dried at 60°C for 5min and finally resuspended in 3uL of nuclease-free water).

Results Summary

Overall, the kit allows the mutagenesis of plasmid vectors of interest, including single or multiple (but relatively adjacent) nucleotide substitutions, deletions and insertions. The protocol is simple but needs some optimization for complex deletions or insertions; generally, it is very fast and straightforward with an average time of less than a week to carry out and confirm a routine mutagenesis. An example is illustrated in the figure below.

Additional Notes

Some components of the kit can be also purchased separately (e.g. Pfu Ultra and DpnI enzymes; XL01-Blue cells). The kits also include a control plasmid with a couple of mutagenesis oligonucleotides, to allow the user to test mutagenesis efficiency. Different formats (10 or 30 reactions) and versions of the kit are available (e.g. including XL10 Gold Ultracompetent cells, which feature an higher transformation efficiency than XL01-Blue cells).

Related Categories

Image Gallery

Colonies of XL01-Blue on an LB agar plate supplemented with the appropriate antibiotic for selection of the plasmid (ampicillin). The results of transformations of two consecutive mutagenesis reactions at the C-terminus of a protein of interest are shown in Panel A. The first one (mut1) causes the substitution of adjacent Lys and Leu to Val and Thr residues respectively, while the second one (mut2) implies the substitution of Phe and Met residues to Ile and a stop codon respectively, causing in this way the C-terminal truncation of the protein. In this case, DNA sequencing has been used for screening; chromatograms of DNA sequences of positive clones for both reactions are reported in Panel B. DNA and protein sequence alignment of the wild-type and mutated sequences is reported in Panel C; only part of the sequences, containing the mutations of interest (KL-->VT in red, FM-->Istop in blue), is shown. Expression test on E.coli BL21 confirm the partial truncation of the protein for the mut2 mutant (see SDS-PAGE on Panel D; bands corresponding to the over-expressed wt and mut2 proteins are high-lighted with red boxes).

Summary

The Good

Easy and efficient way to obtain specific single or multiple mutations.

The Bad

Protocol needs to be optimized in order to obtain large deletions/insertions/substitutions or to mutate regions that require very long mutagenesis primers.

The Bottom Line

The method is smart and simple and could be easily implemented to set up mutagenesis on a large scale. It is worth the expense.

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