competent e coli  (TaKaRa)

Journal: Nucleic Acids Research

Article Title: Systematic screening of archaeal MazF homologs reveals Tth-MazF1, a versatile, sequence-specific ribonuclease from Thermococcus thioreducens

doi: 10.1093/nar/gkag338

Figure Lengend Snippet: Detection of E. coli rRNA modifications by LC-MS/MS following Tth-MazF1 Cleavage. ( A ) A schematic of a Tth-MazF1 digest of a mixture of E. coli rRNA isolated from 70S ribosomes. ( B ) Fractional sequence coverage of detected high-confidence cleavage products in Tth-MazF1 digests of E. coli rRNA. ( C ) Sequence coverage maps of detected high-confidence cleavage products in Tth-MazF1 digests of E. coli rRNA. A complete list and description of the modifications are provided in . Data are a summary of two replicate experiments.

Article Snippet: NiCo21(DE3) E. coli cells (NEB, C2529H) were transformed with 10 ng of each expression plasmid and plated on LB agar containing kanamycin (50 μg/ml) and incubated overnight at 30°C.

Techniques: Liquid Chromatography with Mass Spectroscopy, Isolation, Sequencing

Journal: Nucleic Acids Research

Article Title: Systematic screening of archaeal MazF homologs reveals Tth-MazF1, a versatile, sequence-specific ribonuclease from Thermococcus thioreducens

doi: 10.1093/nar/gkag338

Figure Lengend Snippet: Detection of E. coli rRNA modifications by LC-MS/MS following Tth-MazF1 Cleavage. ( A ) A schematic of a Tth-MazF1 digest of a mixture of E. coli rRNA isolated from 70S ribosomes. ( B ) Fractional sequence coverage of detected high-confidence cleavage products in Tth-MazF1 digests of E. coli rRNA. ( C ) Sequence coverage maps of detected high-confidence cleavage products in Tth-MazF1 digests of E. coli rRNA. A complete list and description of the modifications are provided in . Data are a summary of two replicate experiments.

Article Snippet: Candidate genes were cloned into expression vectors as described in the section “semi-high-throughput expression and purification of maltose-binding protein (MBP)-MazF-6×His proteins.” T7 Express competent E. coli cells (NEB, C1013I) were transformed with 10 ng of each plasmid and plated on LB agar supplemented with kanamycin (50 μg/ml).

Techniques: Liquid Chromatography with Mass Spectroscopy, Isolation, Sequencing