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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 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 membrane retained contaminants migrating between 72 and 118 bp (lanes 4 and 8, respectively). In another series of experiments, the PCR-Script cloning method yielded similar efficiencies and number of transformants, consistently producing greater than an 80% cloning efficiency for PCR fragments prepared by either ethanol precipitation or the StrataPrep kit (data not shown).

Highest Transformation Efficiencies of Ligated DNA

All PCR-Script kits were further improved by adding XL10-Gold Kan ultracompetent cells, which were created by Stratagene to improve the transformation efficiency of ligated DNA into E. coli.7 Ligated DNA molecules transform competent E. coli at a significantly lower efficiency than supercoiled molecules, and large plasmids transform cells less efficiently than small plasmids. This new E. coli host exhibits notably higher transformation efficiencies for ligated DNA and a larger colony size after transformation into XL10-Gold Kan cells; hence, colonies are scored and picked more quickly. Because the blue-white color selection in XL10-Gold Kan cells is unusually vivid, picking the conspicuous colonies is simple.

We tested the transformation efficiencies of the test insert ligated into the pPCR-Script vector into either XL10-Gold Kan or XL1-Blue MRF Kan cells. Four transformations into each of the two cell lines were performed in triplicate. These data confirm that XL10-Gold ultracompetent cells increase transformation efficiencies at least 10-fold over XL1-Blue cells (Figure 2).

Figure 2

Conclusions

Stratagenes PCR-Script cloning kits are outstanding for fast cloning of blunt-end PCR products, no matter what PCR enzyme is used to generate the inserts. Stratagene improved the kits to include the StrataPrep PCR purification kit and XL10-Gold ultracompetent cells. The StrataPrep kit allows PCR products to be rapidly and efficiently purified from excess primers. XL10-Gold Kan ultracompetent cells provide the highest transformation efficiency of ligated DNA and easy-to-score colonies. Finally, these improvements were added to the PCR-Script kit without increasing the price of the kit.

REFERENCES
  1. Bauer, J., et al. (1992) Strategies 5: 62-65.

  2. Pearson, S. and Bauer, J.C. (1996) Strategies 9: 26-27.

  3. Sanchez, T., Zheng, C.-F., and Bauer, J. (1996) Strategies 9: 44-46.

  4. Scott, B., et al. (1994) Strategies 7: 6263.

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

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

  7. Jerpseth, B., Callahan, M., and Greener, A. (1997) Strategies 10: 37-38.

* U.S. Patent Nos. 5,512,468 and 5,707,841 and patents pending.
** U.S. Patent No. 5,545,552 and patents pending.


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