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Enhanced PCR Cloning System

possess 3- to 5-exonuclease activity and generate high-fidelity, blunt-end PCR products.4,5 In cases where PCR products are generated with Taq DNA polymerase or other low-fidelity DNA polymerases, the PCR-Script kits include the reagents necessary to blunt the PCR products for cloning into the PCR-Script vectors.

From purification and cloning of PCR products to transformation of ligated DNA, Stratagene has optimized each step in the PCR-Script cloning kits by incorporating two new products: The StrataPrep PCR purification kit6 makes it easy to prepare PCR products, and XL10-Gold ultracompetent cells7 provide the highest transformation efficiencies of ligated DNA.

Purification of PCR Products

Following PCR amplification, cloning the PCR product requires an ethanol precipitation step to remove excess primers. When multiple samples are processed simultaneously, ethanol precipitation can be cumbersome. To simplify this procedure, all PCR-Script kits now include the StrataPrep PCR purification kit. StrataPrep kits are based on a special DNA-binding fiber matrix in a microspin format,6 which replaces ethanol precipitation of PCR products and allows quick, simultaneous processing of multiple samples. The StrataPrep PCR purification kit removes primers by centrifugal washing; the desired product is eluted in a small volume of buffer or water.

Figure 1

We compared the purity of 0.6- and 1.5-kb PCR products that were prepared using either the StrataPrep kit or the silica-gel-membrane method (Figure 1). Material stained with ethidium bromide and smaller than 100 bp was eliminated from 0.6- and 1.5-kb PCR products purified using the StrataPrep kit (lanes 3 and 7, respectively); whereas, the silica-gel membran
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