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High-Efficiency Site-Directed Mutagenesis of Large Plasmid Vectors

n with an error rate that is significantly lower than conventional thermostable enzymes5 minimizing undesirable second-site mutations.

Improving Mutagenesis of Large Plasmids

In the new QuikChange XL kit, modifications have been made to the QuikChange method to improve the efficiency of mutagenizing large or difficult plasmid vectors. In addition, XL10-Gold ultracompetent cells have been included to ensure the highest transformation efficiencies possible (Figure 1). The transformation efficiency of XL10-Gold cells is 5-fold higher than the transformation efficiency of XL1-Blue cells employed in the standard QuikChange kit6. Moreover, XL10-Gold cells contain the Hte phenotype, which increases transformation efficiency of larger DNA plasmids.

The performance of the QuikChange XL kit is demonstrated in mutagenesis studies employing a 10.1-kb plasmid. In this study, primers were designed to repair a frameshift mutation by inserting the missing nucleotide in the open reading frame (ORF) of the Moloney Murine Leukemia Virus (MMLV) gag-pol fusion protein. QuikChange reactions were performed with components of the standard kit or the QuikChange XL kit. Electrophoresis of Dpn I-digested reaction products showed that bands of the appropriate size (10.1-kb) were only evident in reactions performed with components of the QuikChange XL kit (Figure 2, lane 1). Moreover, transformation of XL10-Gold cells with a QuikChange XL reaction produced 32 colonies. In contrast, no colonies were produced with the standard QuikChange kit (Table 1). Nine out of 10 colonies produced a plasmid with the correct base insertion, as evidenced by DNA sequence analysis, and restoration of gag-pol gene function was verified by assaying reverse transcriptase
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