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Enhanced PCR Cloning System


PCR-Script cloning kits feature new additions

Tim Sanchez Julie Robinson Mary Buchanan
Stratagene

The PCR-Script cloning kits are convenient for cloning PCR fragments generated with any PCR enzyme.1,2 The method is highly efficient, easy, and superior to other available methods.3 Now, Stratagene improves the kits by including both the StrataPrep PCR purification kit, so preparing PCR products is simple, and Epicurian Coli XL10-Gold ultracompetent cells,* so the highest transformation efficiencies of ligated DNA are attained. Furthermore, these innovative additions are available at no extra cost.

The core of these PCR-Script cloning kits is the PCR-Script cloning method,1 in which PCR products are incubated for a 1-hour ligation reaction with one of the Srf I-digested PCR-Script vectors, Srf I restriction enzyme, and T4 DNA ligase. The Srf I enzyme recognizes the rare oligonucleotide sequence 5-GCCCGGGC-3. By including the Srf I enzyme in the ligation reaction, the PCR-Script cloning method maintains high concentrations of digested vector DNA and allows the use of nonphosphorylated, unmodified PCR products. This method ensures a low background of recircularized vector and increased ligation efficiency for blunt-ended DNA fragments by the simultaneous, opposing reactivity of the Srf I enzyme and T4 DNA ligase on nonrecombinant vector DNA. Ligated DNA is then ready to be transformed into competent cells.

The PCR-Script kits, although ideal to use with PCR enzymes such as Pfu and pfuturbo DNA polymerases,,** are versatile and can be adapted for use with any PCR enzyme. Pfu and PfuTurbo DNA polymerases are proofreading enzymes that
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