A 1-g sample of guano was diluted in sterile phosphate-buffered saline, and 3 separate dilutions were filtered through 47-mm, 0.45-μm pore-size, gridded cellulose ester filters.
For each phase of growth, three separate dilutions were tested: 1.2, 6, and 12-fold.
During the primary antibody incubation step, serial dilutions of the egg primary antibody were then prepared in PBS in a separate polypropylene dilution plate (Costar EW-01728-80) before application to the assay plate.
Whereby the solid product that separated upon dilution with water was filtered off and crystallized from the proper solvent.
The reaction mixture was then left overnight at room temperature, whereby the solid product that separated upon dilution with water was filtered off and crystallized from ethanol as orange crystals, yield 22%, 0.77 g, m.p. 224 225 °C.
Separate standard dilutions were prepared for medium- and cell extract-conditions respectively, because of the samples' different buffer conditions.
Each antibody was incubated in separate membranes (1 : 1000 dilution), following the instructions of the supplier.
Sequences of porcine and bacterial primers as well as PCR efficiencies (based on three separate 5-fold dilution series of pooled cDNA samples, run on a separate qPCR chip), Tm, and amplicon lengths can be found in Additional file 2: Table S2.
In a separate experiment, the dilution effect of plant extract on AChE activity was measured after gentle mixing 110 μl of (100 μl enzyme:10 μl plant extract) with 890 μl of the same above mixture except that 90 μl plant extract was replaced with 90 μl DMSO (solvent).
