Ecos competent escherichia coli jm109

Plasmid cloning vectors with spike-in sequence inserts were transformed into ECOS Competent Escherichia coli JM109 (Nippon Gene, Toiya, Japan) following the manufacturer's instructions. Plasmid DNA was extracted from overnight liquid cultures using the QIAGEN Plasmid Midi Kit. Plasmid DNA was then linearized using the following single-cutting restriction enzymes, according to the manufacturer's instructions: BpmI (New England Biolabs) for spike-ins Ec5001, Ec5002, Ec5005, Ec5502 and Ga5501; BsaI-HF (New England Biolabs) for Ec5003, Ec5004, Ec6001, Bv5501, Ca5501 and Tb5501; and ScaI (TaKaRa Bio) for Ec5501. Linearized plasmid DNA was purified using the Agencourt AMPure XP system (Beckman Coulter) and size and integrity were verified by electrophoresis using the Bioanalyzer 2100 with a DNA 12000 Kit (Agilent). DNA concentrations were determined with a high-sensitivity Quant-iT dsDNA Assay Kit (Invitrogen) using a Qubit Fluorometer 3.0 (Life Technologies). Plasmid DNA was diluted to 10 ng/μl in Tris-EDTA (TE) buffer (pH 8.0) and distributed in single-use aliquots stored at −80°C. Spike-in sequences were verified by Sanger sequencing (see Supplementary Data for details) and experimentally determined sequences were in all cases in agreement with designed sequences. Spike-in standard mixes were prepared based on estimated copy numbers and stored in TE buffer at −20°C until use.