Experiment SRX078307 Download

TitleWhole Genome Sequencing for JM221
AliasJM221 sequencing
Design descriptionFor the 6 non-outbreak O104 strains and 5 reference isolates, we grew bacterial cultures overnight from a population in 50 ml of Luria broth, minimizing the number of passages of each strain. Genomic DNA was isolated according to standard methods (Z Ge, et al. Clayton CL, Mobley HLT, eds. Helicobacter pylori Protocols. Totowa: Humana Press Inc., 145-52 (1992)). Briefly, bacterial cells were concentrated by centrifugation, washed and suspended in isolation buffer (0.15 M Tris, 0.1 M EDTA, pH 8.0). Sodium dodecyl sulfate was then added to 1% (vol/vol) final concentration and allowed to incubate for 1 hour at 55?C or until the solution cleared. Two volumes of phenol:chloroform:isoamylalcohol (25:24:1) were added and briefly mixed by vortex. The resulting solution was separated by centrifugation at 12,000xg for 15 minutes at 4?C. The aqueous layer (top) was removed to a new tube and mixed with two volumes of chloroform. The mixture was separated by centrifugation at 12,000xg for 15 minutes at 4?C and the aqueous layer (top) was moved to a clean tube. The aqueous layer was then extracted with at least 10 volumes of ice-cold ethanol and the precipitated DNA was spooled out of the mixture and suspended in ultrapure water. The purified mixture was further digested with RNase at 37?C and re-precipitated with 0.1 volumes of 3M NaOAc sodium acetate and 10 volumes of ice-cold ethanol. The pellet was allowed to air dry and then dissolved in a minimal volume of nuclease-free water (Ambion). The quantity and quality of genomic DNA was verified by gel electrophoresis, spectroscopy and picogreen assay.
Referencesno content
OrganismsEscherichia coli
SubmitterPacific Biosciences
InstrumentPacBio RS
Library nameJM221 8kb SMRTbell
Library strategyWGS
Library sourceGENOMIC
Library selectionRANDOM
Library layoutSINGLE
Library construction protocolno content
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Linksno content