Nanodrop 2000 uv vis spectrometer

The hydrodynamic sizes and zeta potentials were measured on a Nano-ZS Zetasizer dynamic light scattering (DLS) instrument (Malvern Instruments Ltd., Malvern, UK). The microstructure of the sample was measured using a field emission scanning electron microscope (SEM, ZEISS-ULTRA55; Hitachi Ltd., Tokyo, Japan) and the particle diameters were calculated using Nano Measurer 1.2 software. IR analysis and UV analysis were carried out on Nicolet 5700 FTIR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) using the KBr-disk method and Nanodrop 2000 UV–vis spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), respectively.

The abundance of the mcrA-genes in the microbial communities was quantified by real-time qPCR using an Mx3005P (Agilent, Santa Clara, USA). DNA concentrations in the cell extracts were measured by NanoDrop 2000 UV–Vis Spectrometer (Thermo Scientific, Waltham, USA) followed by individual dilution to reach a final concentration of approximately 5 ng/approach. Diluted samples were mixed with 5 µL of SybrGreen Mastermix (Kapa, Wilmington, USA), 0.2 µL ROX Low reference Dye (Kapa, Wilmington, USA) and 0.25 µL of the two primers (Eurofins, Ebersberg, Germany), namely mlas and mcrA-rev (Steinberg and Regan 2008 (link)) to give a final total volume of 10 µL. The mixture was pipetted into a low profile 96 well plate (Thermo Scientific, Erlangen, Germany), sealed with Ultra Clear Cap Strips (Thermo Scientific, Erlangen, Germany), and centrifuged for 5 min at 500×g. qPCR conditions were as follows: 3 min at 95 °C, then 40 cycles of denaturation at 95 °C for 30 s, annealing at 62 °C for 45 s and extension at 72 °C for 30 s. The final cycle was 95 °C for 15 s, 55 °C for 30 s and 95 °C for 30 s. For quantification a plasmid standard was prepared by cloning and ligation of a mcrA sequence by CloneJET PCR Cloning Kit (Thermo Scientific, Erlangen, Germany). Correctness of the DNA insert was verified by sequencing (Microsynth AG, Balgach, Switzerland).

Total RNA was isolated from cultured cells homogenized in QIAzol Lysis Reagent (Qiagen, Germany) using a single‐step guanidine isothiocyanate/phenol/chloroform‐based extraction technique. RNA concentration and quality were measured using a NanoDrop 2000 UV‐Vis Spectrometer (Thermo Fisher Scientific, Wilmington, DE). Using RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific), 1000 ng of RNA were reverse‐transcribed to cDNA. For quantitative polymerase chain reaction (qPCR) analysis, CFX96 Real‐Time PCR Detection System (BioRad, Hercules, CA) was employed using Kapa Sybr Fast qPCR Master Mix (2x) Kit (Kapa Biosystems, Wilmington, MA). RNA expression of target genes relative to glyceraldehyde‐3‐phosphate dehydrogenase was quantified by ΔΔCT method. Statistical analysis was performed in GraphPad Prism 6.