( ...)Highest Possible Transformation Efficiencies for High-Throughput ,,, Applications

Convenient, time-saving 96Pack Gold competent cells streamline transformation procedures

Julie Bonnardel Ronda Allen Bruce Jerpseth Sean Banks

Stratagene

The new 96Pack Gold competent cells* from Stratagene feature a convenient, high-throughput format that saves time without compromising quality. These cells feature exceptionally high transformation efficiencies, fast growth, blue-white color screening of recombinants, and high-quality plasmid miniprep DNA. Simplified transformations, fast results, and ease of use make these cells ideal for all high-throughput cloning applications. For transformations of supercoiled or ligated DNA, we compared the performance of 96Pack Gold cells with a competitors 96-well-format competent cells and found that our cells consistently yielded higher transformation efficiencies.

With continuing advances in molecular biology technology, high-throughput methods have become increasingly important. With the 96Pack Gold competent cells, Stratagene has combined convenience with superior quality to ensure the best possible performance in a high-throughput format.

Combining Highest Efficiency with a High-Throughput Format

Stratagenes Hte phenotype, introduced in 1997, represented a breakthrough in competent-cell technology. Until that time, the best possible transformation efficiency was just higher than 5 x 10⁹ transformants per microgram of supercoiled pUC18 vector (2.7 kb); efficiencies for ligated DNA were lower as expected. With the advent of the Hte phenotype, transformation efficiencies for ligated DNA increased 25-fold. This phenotype allows competent cells to better accept ligated or larger DNA plasmids, which are known to transform less efficiently than small plasmids on a per molecule basis. Due to exceptional performance, we have found that competent cells with the Hte phenotype are the best choice for any cloning experiment that demands a high efficiency of retrieving transformants from ligation reactions, such as constructing plasmid libraries and PCR cloning. The Hte phenotype helps 96Pack Gold cells fulfill the need for streamlined transformation procedures and superior performance.

In the basic high-throughput procedure, DNA is added to each well of 96Pack Gold cells with a multichannel pipettor. The microtiter plate is chilled and then subjected to heat shock, which is simply performed with either a thermocycler, a heating block, or a conventional water bath. Growth medium is added to the wells. After incubations, transformants are selected with the appropriate antibiotic medium. This simple protocol saves time and reagents, while providing the highest possible transformation efficiencies.

Engineered to Achieve the Highest Performance

96Pack Gold competent cells carry the Hte mutation, which confers the capacity to transform large and ligated DNA molecules at a higher efficiency. The 96Pack Gold strain is endonuclease-deficient (endA1), which greatly improves the quality of plasmid miniprep DNA. These cells are recombination-deficient (recA), ensuring the stability of inserted DNA. With the lacI^qZDM15 gene on the F episome, 96Pack Gold competent cells allow bluewhite color screening of recombinant plasmids. In addition, this cell strain is restriction-minus, a feature that increases the efficiency of introducing eukaryotic DNA into E. coli and increases the size and representation of libraries constructed with methylated or hemimethylated DNA. Finally, 96Pack Gold cells grow at a fast rate, resulting in the formation of larger, easier-to-select colonies (Figure 1).

Superior Performance

We compared the transformation efficiency of 96Pack Gold cells with MultiShot cells (Invitrogen) (Figure 2). To test efficiency with supercoiled DNA, we added 1 pg and 10 pg of the pUC plasmid to either 96Pack Gold or MultiShot cells. To test efficiency for ligated DNA, we transformed 96Pack Gold cells and MultiShot cells with 2.3 ng of a ligated construct, the pCMV-Script plasmid with the LacZ gene insert (approximately 4.9 kb). For each experiment, we followed the manufacturers protocols. As can be seen, 96Pack Gold competent cell transformation efficiency outperformed the competitors product with both supercoiled and ligated DNA.

Conclusions

Stratagenes high-efficiency 96Pack Gold competent cells, provided in a high-throughput format, are the superior choice for PCR cloning, restriction enzyme cloning, and ligase-independent cloning. In comparison transformations of either supercoiled or ligated DNA, 96Pack Gold chemically competent cells yielded higher efficiencies than another 96-well-format product. We confirmed that these cells streamline transformation procedures, and outperform other commercially available cells.

REFERENCE

1. Jerpseth, B., et al. (1997) Strategies 10: 37-38.

Genotype

XL10-Gold strain: Tet^r D(mcrA)183 D(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte [F proAB lacI ^qZDM15 Tn10 (Tet^r) Amy Cam^r]

* U.S. Patent Nos. 5,512,468 and 5,707,841 and patents pending

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