The best approach found to minimize incubation time and reduce errors was to select the minimum sediment thickness necessary to include the entire depth distribution of nitrification denitrification for a particular sediment system.
Since a short incubation time was desirable to minimize cell death and damage to short-lived macromolecules, this minimal incubation time is considered an important advantage.
The five-week experimental incubation time minimized the risk of invasion by non-target animals, prevented the complete consumption of eelgrass by the grazers, and permitted major changes in animal (one to two grazer generations) and plant community development and in surface sediment characteristics [45], [48], [50].
We minimized the variability of incubation time by first mixing diluted sera (1/10) with a fixed quantity of monoclonal antibody in a normal non-coated plate and then transferring the mixture to a precoated plate and incubating at 37°C for one hour, with gentle shaking.
Under the circumstances, specific steps were taken to minimize this unwanted situation and may include longer antibody incubation time (overnight at 4°C) followed by more stringent wash (twice in lysis buffer and three times in HEPES buffer).
Preliminary experiments were performed to identify the optimal concentration of dexamethasone and the incubation time that achieved the best compromise between minimizing spontaneous cell death and maximizing dexamethasone-induced killing (Supplementary Figure 1a).
At the same time, shortened incubation time for impregnation and activated molecular movements may minimize precipitate formation.
To minimize non-specific binding and uptake of the aptamer nanostructures, stringently controlled conditions were used: (i) short incubation time (1 h), (ii) relatively low concentration of aptamer aptamer complexes (~0.5 µM), and (iii) co-incubation with 1 mg/ml of tRNA + ssDNA (0.5 mg/ml each).
This incubation time was pre-determined to be optimal for detecting maximal expression of the analytes being measured while minimizing loss due to in vitro consumption (data not shown).
A 120 min RT incubation time and an extension time of 1.5 2 min/kb of template DNA were suggested as suitable to minimize recombination in most long RT-PCR [24].
