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Wet cells were suspended (0.5 mg/mL) in Y-per yeast or B-per bacteria extraction protein reagent (Pierce Biotechnology, Rockford, IL, USA) in a 2-mL Eppendorf tube and incubated on a turning table at room temperature for 50 minutes.
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To determine the absolute number of bacteria per extraction, we extracted infected fly hemolymph and used fluorescence microscopy to calculate the concentration of Spiroplasma cells stained using SYTO9 (as described above).
Samplings were done at 4 locations on the Modesto dairy and 3 locations on the Sonoma dairy (Figure 1) and the aerosol wash-water was pooled from all units and locations for characterization of aerobic bacteria and extraction of environmental DNA.
Chromosomal DNA was prepared using the MiniBEST Bacteria DNA Extraction Kit Version 2.0.
While, leaching by the original bacteria, the extraction of total iron was 85.8% after 15 days.
Briefly, the DNeasy Blood & Tissue Kit: gram positive bacteria DNA extraction protocol was used with some modifications.
When leaching by the original bacteria, the extraction of total arsenic was only 46.5% after 12 days.
Then 4 mL cultures were centrifuged to obtain cell pellets for C. cellulolyticum genomic DNA isolation using a TIANamp Bacteria DNA Extraction Kit (Tiangen Biotech, Beijing, China).
Then we attempted to apply the same method to recover the adherent population of the TIGR4 strain, but we were unable to retrieve enough TIGR4 bacteria for extraction of sufficient amounts of RNA.
Chromosomal DNA of screened bacteria was extracted using CinnaPure TM DNA extraction kit (Cinnagen, Iran) according to manufacturer's instructions.
Finally, all samples had an anti-bacterial effect against different types of bacteria and the extraction of silver ions from them followed a diffusion-controlled mechanism, which could demonstrate their ability to treat bone infection.
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