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XL10-Gold Cells Supplied in New Single-Reaction Format


solopack Gold cells reduce the time and effort needed for transformationswithout compromise!

Anna Waesche Jonathan Goodwin Amy Duncan Bruce Jerpseth Alan Greener
Stratagene

Stratagene introduces competent cells from the XL10-Gold strain* in an easy-to-use, single-reaction format that yield the supercompetent efficiency of 1 x 10 9 transformants/g of supercoiled DNA or the competent efficiency of 1 x 10 8 transformants/g of supercoiled DNA. The new Epicurian Coli SoloPack Gold supercompetent and competent cells exhibit the Hte phenotype for high transformation efficiency and exhibit robust growth to produce large colonies. The SoloPack format saves time by providing a convenient transformation format.

Transformation of competent E. coli cells is crucial for cloning a gene, expressing a protein, or constructing a plasmid library. In many of these instances, transformation is the rate-limiting step. Working with an E. coli strain that efficiently transforms ligated DNA in a convenient format is, therefore, desirable. Stratagene developed the XL10-Gold strain, containing the Hte phenotype,** to produce the highest transformation efficiencies for ligated DNA. This phenotype is observed in cells that carry an uncharacterized mutation that has been shown to improve the efficiency of transforming ligated plasmids and large DNA molecules relative to strains of equal competency (as measured by supercoiled pUC DNA transformation).1,2

In any transformation reaction, competent cells must be thawed and aliquotted into Falcon 2059 polypropylene reaction tubes. To speed up the transformation process, we designed the SoloPack format so that the entire transformation reaction, including post heat-shock expression, is performed in a single tube. Th e SoloPack single-tube, single-reaction format is available at the supercompetent efficiency of 1 x 109 transformants/g of supercoiled DNA and the competent efficiency of 1 x 108 transformants/g of supercoiled DNA.

Although this new format is convenient, it is especially important to maintain high transformation efficiency and not compromise the outcome of the cloning experiment. Hence, we chose our most efficient competent cell strain, XL10-Gold, and created SoloPack Gold supercompetent cells and SoloPack Gold competent cells. Use the supercompetent efficiency (1 x 109 transformants/g of supercoiled DNA) for PCR cloning, restriction enzyme cloning, the ligase-independent cloning (LIC) method,3 and expression vector construction. Or, as an economical alternative, use the competent efficiency of 1 x 108 transformants/g of supercoiled DNA when high efficiency is not required. For plasmid library construction and other demanding experiments where optimal transformation efficiency is critical, Stratagene provides the XL10-Gold ultracompetent cells in our standard-tube format with efficiency greater than 5 x 109 transformants/g of supercoiled DNA.

Transformation

We tested the transformation efficiency of 1 x 109 transformants/g of supercoiled DNA for the SoloPack Gold supercompetent cells: The cells were thawed on ice, 1 l of XL10-Gold b-mercaptoethanol mix was added, and the cells were incubated on ice for 10 minutes. Next, 10 pg of pUC DNA was added to the cells and they were incubated on ice for an additional 20 minutes. The cells were then heat pulsed at different temperatures for varying lengths of time to optimize efficiency. After the heat pulse, 0.15 ml of NZY+ outgrowth media was added, and the cells were shaken in the reaction tubes for 60 minutes prior to plating. Our results showed t hat a heat pulse of 54C for 60 seconds was optimal to achieve an efficiency of 1 x 109 transformants/g of supercoiled DNA in the single-tube format.

The XL10-Gold strain exhibits a faster growth rate and produces larger colonies than other commonly used hosts like DH5a and XL2-Blue. To quantitate this difference, we performed a standard growth curve for these strains harboring the pBluescript plasmid. Results in Figure 1 show that XL10-Gold cells grew demonstrably faster than DH5a and XL2-Blue cells.

Figure 1

Conclusions

Stratagene now provides SoloPack Gold supercompetent cells (1 x 109 transformants/g supercoiled DNA) and competent cells (1 x 108 transformants/g supercoiled DNA) created from our XL10-Gold strain in a handy single-reaction format. The XL10-Gold strain exhibits the Hte phenotype for high transformation efficiency and provides robust growth to produce larger colonies than other commonly used hosts. Combine the XL10-Gold strain with the SoloPack format, and streamline the transformation process while retaining high efficiency.

Genotype

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

The XL10-Gold strain is tetracycline and chloramphenicol resistant.

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

  2. Jerpseth, M., et al. (1998) Strategies 11: 3-4.

  3. Aslanidis, C. and de Jong, P.J. (1990) Nucleic Acids Res. 18: 6069-6074.

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


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