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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 ctor DNA (50 ng in 0.3 l; purified using the StrataPrep plasmid miniprep kit3), QuikChange 10X reaction buffer (5 l), primers (1 l of each at a concentration of 125 ng/l), and water (40.7 l) were combined and placed in Block 1 of a RoboCycler temperature cycler, at 95C for 2 minutes. The thermal cycler was turned off to allow the samples to cool slowly to room temperature. The reaction mixture was placed on ice while adding 1 l of PfuTurbo DNA polymerase (2.5 U), QuikChange dNTP mix (1 l), and a drop of mineral oil. The reaction mixture was then placed in Block 3 of the cycler at room temperature, and the unit was turned on so the block could slowly heat to 68C. The reaction mixture was incubated at this temperature for 5 minutes followed by 18 cycles of 95C for 1 minute, 55C for 1.5 minutes, and 68C for 12 minutes. At the end of temperature cycling, 10 U (1 l) of Dpn I restriction enzyme were added, and the reaction mixture was incubated at 37C for 1 hour. Following Dpn I digestion of parental DNA, 1- and 4-l volumes of the reaction mixture were used to transform XL1-Blue supercompetent cells. The transformed cells were spread onto LB/ampicillin plates, as described in the QuikChange manual. Random ampicillin-resistant colonies were inoculated into tubes containing 1.5 ml of LB media and 100 g/ml of ampicillin and incubated with shaking for 12 hours at 37C. Plasmid DNA was purified from the cultures using the StrataPrep plasmid miniprep kit.3 Plasmid preparations were digested separately with Stratagenes Bgl II, Pst I, and Xho I restriction enzymes. Plasmid DNA was digested with all 3 enzymes in 9 out of 12 randomly chosen clones. The Bgl II, Pst I, an d Xho I restriction digestion patterns for seven of the clones are shown in Figure 2, indicating that the appropriate restriction sites were successfully introduced into the plasmid vector.

Fig.2

Conclusions

These results demonstrate that a 28-base insertion, encoding three unique restriction enzyme sites, was effectively introduced into a plasmid vector using the QuikChange mutagenesis kit. Mutagenesis reactions and transformation can be completed in less than one day so putative positive clones may be examined the following day. And, because high-fidelity PfuTurbo DNA polymerase is used, random mutations are minimized, rendering this the preferred method for efficient mutagenesis.

REFERENCES
  1. Papworth, C., Bauer, J.C., and Braman, J. (1996) Strategies 9: 3-4.

  2. Hogrefe, H., et al. (1997) Strategies 10: 93-96.

  3. Braman, J. and Basehore, S. (1997) Strategies 10: 84-86.

* U.S. Patent No. 5,789,166 and patents pending
** U.S. Patent Nos. 5,545,552 and 5,866,395 and patents pending


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