( We compared the ...)New Competent Cells for Highest Transformation Efficiencies
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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
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