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Efficiently Insert Unique Restriction Sites into Plasmid Vectors


Perform insertion mutagenesis in less than one day

Lisa Breister Jeff Braman
Stratagene

A 28-base insertion was introduced into a plasmid vector using the QuikChange site-directed mutagenesis kit.* The insertion created three unique restriction enzyme digestion sites. Plasmid DNA isolated from 9 out of 12 randomly chosen transformed clones were digested with all 3 restriction enzymes, as judged by gel electrophoresis.

With Stratagenes QuikChange kit,1 performing site-directed mutagenesis is simple, accurate, and efficient. This method permits single and multiple bases to be easily deleted and inserted into any double-stranded plasmid vector, eliminating the need for single-stranded DNA rescue and subcloning into specialized vectors. The kit relies on high-fidelity PfuTurbo DNA polymerase,** which accurately replicates plasmid DNA, thereby minimizing second-site mutations.2 These features make the QuikChange procedure the method of choice for most mutagenesis projects.

Here we describe insertion mutagenesis of a plasmid vector, which resulted in the creation of three unique restriction enzyme recognition sites.

Site-Directed Mutagenesis

To introduce restriction enzyme recognition sites into the plasmid vector, homologous oligonucleotides (49 bases long) were constructed (Figure 1). The oligonucleotides contained ten, 5 terminal bases and eleven, 3 terminal bases homologous to the 6-kb vector. The 28 interior nucleotides destined for insertion into the vector included restriction enzyme recognition sites for Bgl II, Pst I, and Xho I.

Fig.1

Modified pBK388 ve
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