Following optimisation, reaction mixes (100 μl) were set up as follows -10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 200 μM (each) dATP, dCTP, dGTP and dTTP; 1.25 U ofollows -10olymMrase (Amplitaq; Perkin Elmer), 0.1 μM (each) of the each set of pHimers (exoA & oprL) (Table 1) and 4 μl of DNA template.
Following optimisation of the reaction conditions, conjugates for subsequent experiments were synthesised with eightfold excess of linker 4 in step 1.
Following optimisation of parameters for the introduction of FMDV RNA into BHK cells, a standard protocol was adopted as described in Material and Methods.
Following optimisation of the conditions for maintaining dispersed growth of EcadAb ES cells in shake flasks we assessed fed-batch culture of the cells over 15 d (Figure 3F).
Following optimisation of the conditions required for efficient sGAG digestion, the presence of sulphated species in the UBH was decreased (Figure 2C) and a reduction of polysaccharide chains shown by decreased absorbance of purified GAGs separated using an anion exchange column (Figure 2D).
Following optimisation, all microscope settings were rigorously adhered to.
Following optimisation, selected cultures were carried out using animal-free reagents.
Following optimisation of tissue thickness, cryosections of 6 μm thickness were found to be effective for LMD.
Following optimisation of the antibody for frozen tissue, the sections were incubated with 1 : 150 dilution of primary antibody (Novus Biologicals) overnight at 4°C.
Following optimisation, cells were seeded at 3.5 × 10 cells/well in 24-well plates or 1.75 × 10 cells/well into 6-well plates.
Following optimisation, fluorescent dot blot analysis of transfection supernatants was shown to be a rapid method for analysing protein expression yielding similar results as western blot analysis.
