The effect of ignition delay on the exhaust emissions of these pollutants was isolated by adding the ignition promoting molecule 2-ethylhexyl nitrate to some of the fuel samples in closely specified concentrations, so as to equalise the ignition delay for the relevant fuel samples.
Chromatin was isolated by adding lysis buffer, followed by disruption with a Dounce homogenizer (cells).
Genomic DNA was isolated by adding an equal amount of isopropanol, mixing and subsequent centrifugation for 20 min at 6000 g.
Circulating RNA was isolated by adding to 250 µl of serum an equal volume of Tri-Reagent BD (Sigma, St Louis, MO), the phase lock gel (Eppendorf) was used to improve RNA recovery.
Subsequently, RNA was isolated by adding 790 μL of buffer containing absolute ethanol, along with passage through a purification column.
Escherichia coli was isolated by adding 1 g faeces to 9 ml Gram-negative broth (Becton & Dickinson, Franklin Lakes, USA).
After the tumour cells had been collected into a 0.5-ml reaction tube, DNA was isolated by adding 50 μl of proteinase K digestion buffer.
After clearing cellular debris by spinning at 14,000 g for 10 min at 4°C, DNA was isolated by adding an equal volume of phenol chloroform:isoamylalcohol (25:24:1), vortexing, and spinning at 12,000 g for 5 min at room temperature.
Prior to qPCR, RNA from cells was isolated by adding TrizolTM reagent (Invitrogen, Karlsruhe, Germany) directly into the wells, and pipetting the lysed cells up and down 2 3 times.
The RNA fraction was isolated by adding 0.75 ml of Trizol LS reagent (Invitrogen, Carlsbad, CA) to 0.25 ml plasma, followed by incubation at room temperature for 5 min. To this solution, 0.2 ml of chloroform was added; after vigorous mixing, it was further incubated for 5 minutes at room temperature, before centrifugation at 15,000 x g for 15 min at 4°C, to obtain the RNA aqueous phase.
