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Enhanced PCR Cloning System

e 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 improv
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