Perform insertion mutagenesis in less than one day
Lisa Breister Jeff Braman
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.
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.
Modified pBK388 ve