Takara Stellar Electrocompetent Cells \10 Transformations

Stellar Electrocompetent Cells are anE. coliHST08 strain that provides high transformation efficiency, good reproducibility, and blue/white screening when paired with pUC plasmid vectors. These cells are specially made for transformation using the electroporation method. Stellar Electrocompetent Cells lack the gene cluster which digests foreign methylated DNA (mrr-hsdRMS-mcrBCandmcrA), and are therefore useful for cloning of methylated DNA, in addition to construction of genomic libraries and longer-length genomic libraries. A pUC19 vector is provided as well as SOC Medium.

The high efficiency of Stellar Competent Cells was especially noticeable in the transformation of 10 kb and 20 kb plasmids.Using DNA plasmids of 2 kb (100 pg), 10 kb (1 ng), and 20 kb (1 ng), each strain of competent cells was transformed and plated on ampicillin-containing LB agar plates. Transformation efficiencies were determined based on the colony counts obtained.

The difference in transformation efficiencies of the competent cells was particularly prominent when cloning 20 kb fragments.Ligation reactions were set up with either 100 ng of a 2 kb, Hind III-cut DNA fragment + 50 ng of Hind III/BAP-cut pUC118 vector, or 75 ng of a 20 kb, Hind III-cut DNA fragment + 25 ng of Hind III/BAP-cut pUC118 vector. All reactions were performed at 16℃ for 6 hours using the Takara DNA Ligation Kit (Cat# 6023 or 6024). Each strain of competent cells was transformed using portions of these reaction mixtures and plated on ampicillin-containing LB agar (+ X-gal). Transformation efficiencies were determined based on the white colony counts obtained.

Stellar colonies transformed with larger plasmid clearly showed a faster growth rate.DNA plasmids of 2 kb (Panel A) and 10 kb (Panel B) were each used to transform Stellar and DH10B competent cells with similar genetic characteristics, including methylation requiring restriction. This experiment used the same method as in Figure 1. Photographs of colonies on agar were taken after 15 hours of incubation.