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New Competent Cells for Highest Transformation Efficiencies

Up to 25-fold higher transformation efficiency of ligated DNA

Bruce Jerpseth Marie Callahan Alan Greener
Stratagene Cloning Systems, Inc.

Stratagenes new, high-efficiency competent E. coli cell line significantly improves the transformation efficiency of ligated DNA. Epicurian Coli XL10-Gold ultracompetent cells* yielded transformation efficiencies for ligated DNA that were up to 25-fold higher than other commercially available cell lines. XL10-Gold cells are the host cells of choice for any cloning experiment that requires a high efficiency of retrieving transformants from ligation reactions, such as constructing plasmid libraries and PCR cloning.

The ability to efficiently introduce DNA into E. coli by chemical transformation and electroporation is a key step for many procedures, such as generating plasmid libraries, cloning large DNA fragments and cloning PCR products.1 The cells that have set the industry standard for these types of procedures are Stratagenes ultracompetent cells, which yield greater than 5 x 109 transformants/g of supercoiled pUC18 vector (2.7 kb). However, these transformation efficiencies are typically calculated using very small amounts of supercoiled pUC18 vector DNA. Ligated DNA molecules are known to transform cells at a significantly lower efficiency than supercoiled molecules, and large plasmids transform cells less efficiently than small plasmids. This observation has particular impact for researchers making plasmid libraries: the total number and size distribution of primary transformants is key to finding the gene of interest and affects how easily full-length clones can be located. The bias against transforming larger molecules influences the ability to retrieve full-length cDNA clones. For example, many plasmid library vectors, including two-hybrid vectors and eukaryotic expression vectors, are large to begin with, making this size bias more acute. Plasmid cDNA libraries constructed in current E. coli hosts have limited numbers of primary isolates and do not accurately reflect the cDNAs that are present.

Stratagene has endeavored to maximize the size of plasmid libraries and minimize the size bias when constructing plasmid libraries by improving the efficiency with which ligated DNA can transform E. coli. These efforts have resulted in the creation of XL10-Gold ultracompetent cells, a new E. coli host that exhibits notably higher transformation efficiency for ligated DNA molecules. The ability to better accept ligated, or larger, DNA molecules means that a plasmid library constructed in XL10-Gold cells will more faithfully represent the population of cDNA molecules.

Transformation of a Large Supercoiled Plasmid

Host

pRK2013

pUC18

efficiency/mg DNA

Fold difference

efficiency/mg DNA

Fold difference

DH10B
cells

5 x 102

1

1 x 109

1

XL2-Blue cells

4 x 103

8

5 x 109

5

XL10-Gold cells

4 x 104

80

5 x 109

5

A 25-kb supercoiled plasmid, pRK2013, was transformed into three different competent cell lines (figure 1): XL10-Gold, XL2-Blue cells and DH10B cells.** The pRK2013 plasmid was selected to simulate the large DNA molecules that exist when the transformation substrate is a ligated molecule. These data show that XL10-Gold cells were up to 80-fold more efficient than DH10B and 10-fold more efficient than XL2-Blue cells at transforming this large plasmid. The efficiency with which supercoiled pUC18 transformed was the same for both XL10-Gold and XL2-Blue cells and 5-fold better than for DH10B cells.

Therefore, XL10-Gold ultracompetent cells retain their high efficiency when transforming pUC18 vector DNA and are significantly better at transforming the large supercoiled plasmid than either XL2-Blue or DH10B cells.

Transformation of Ligated DNA

DH5a cells

XL10-Gold cells

Colony Number (average of 3 experiments)

261

7250

pUC18 Efficiency (cfu/mg)

1 x 109

5 x 109

A second assay was performed to determine the relative efficiency of transforming XL10-Gold cells with a ligated DNA molecule. The pCAL-n-EK vector (8.0 kb) from Stratagenes Affinity LIC cloning kit was digested and prepared for the ligation-independent cloning (LIC) reaction using standard conditions.2 The kanamycin-resistance gene was annealed to the pCAL-n-EK vector. An aliquot of the annealed mix was used to transform both XL10-Gold cells and DH5a** cells (table 1). XL10-Gold ultracompetent cells yielded 27-fold more kanamycin-resistant transformants than DH5a cells. The pUC18 vector was used as the supercoiled control plasmid for this experiment, transforming XL10-Gold cells with a 5-fold higher transformation efficiency than DH5a cells.** These data support our finding that XL10-Gold cells have a significantly improved ability to transform larger (ligated or LIC-annealed) DNA molecules.

Transformation of cDNA Libraries

pUC18

pCMV-Script Library

pAD-GAL4 Library

Host


cfu/
mg

fold
difference

total
colonies

fold
difference

total colonies

fold
difference

DH10B cells

1 x 109

1

2.3 x 104

1

2 x 103

1

XL2-Blue cells

5 x 109

5

8 x 104

3.5

1 x 104

5

XL10-Gold cells

5 x 109

5

4 x 105

17.4

5 x 104

25

We compared the relative efficiencies of XL10-Gold and DH10B cells for transforming a cDNA library. In order to explicitly address both the overall efficiency and the potential size bias, two plasmid vectors were used. Stratagenes pcmv-script vector (4.2 kb) was digested with Xho I and EcoR I; Stratagenes two-hybrid pAD-GAL4 vector (7.2 kb) was also digested with the same two enzymes. A mouse brain cDNA library was prepared from 5 g poly(A)+ mRNA using Stratagenes cDNA Synthesis Kit. The ligations were performed in a 15-l reaction volume using 30 ng of vector and 10 ng of library cDNA. Of this ligation reaction, 1 l was transformed into both XL10-Gold and DH10B cells (figure 2). As an internal standard, 100 pg of supercoiled pUC18 vector DNA was used to monitor transformation efficiency.

These data show that XL10-Gold cells yielded 25-fold more colonies than DH10B cells for the pAD-GAL4 vector library and 20-fold more colonies for the pCMV-Script vector library. The increase in colony number for XL10-Gold ultracompetent cells provides compelling evidence that XL10-Gold cells are more efficient for the transformation of large ligated molecules.

Conclusions

XL10-Gold cells efficiently transform large DNA molecules and, therefore, represent a significant breakthrough in competent cell technology. XL10-Gold ultracompetent cells transform ligated DNA with higher efficiency than XL2-Blue, DH5a and DH10B cells. This improved transformation efficiency is essential for constructing plasmid libraries since the host strain affects both the size of the primary library (in total numbers) and the ability to retrieve a representative population of molecules. For any experiments requiring high efficiency ultracompetent XL10-Gol d cells are the gold standard.

Acknowledgments

The authors wish to thank Quinn Lu, Barry Neiditch, Peter Vaillancourt, Denise Wyborski and Alexandra Bernardino.

  1. Hannahan, D., and Bloom, F.R. (1996) In Escherichia coli and Salmonella, 2nd ed. (Neidhardt, et al., eds.), pp.2449-2459. American Society of Microbiology, Washington, D.C.

  2. Wyborski, D.L., Bauer, J.C., McGowan, B., Sorge, J.A., and Vaillancourt, P. (1997) Strategies 10: 15-18.


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