Amplification of the latter sequence was used as a positive control for successful DNA extraction from the samples.
For Cynachum daltonii, populations were sampled on all seven islands from which it has been recorded but DNA extraction from the samples from Santiago and São Vicente failed.
DNA extraction from the samples was performed using commercially available isolation kits (DNeasy Kit; Qiagen, Germantown, MD) according to the manufacturer-suggested protocols.
Through the use of solid phase extraction (SPE), STX243 was extracted from the samples under vacuum using Oasis HLB Extraction Cartridges (Waters, Elstree, Herts, UK).
Because SSID MS methodology cannot compensate for non-quantitative extraction from the sample, aggressive conditions are required for solid liquid extraction of strongly adsorbed species, for example MBT.
Although SSID MS methodology can correct most of the systematic errors that occur in the analysis, it can not compensate for non-quantitative extraction from the sample or for species re-conformation in the sample preparation process [ 6– 11].
The T-RFLP data were highly reproducible based on replicate extractions from the fecal samples and restriction digestions with three different restriction enzymes (data not shown).
Edgcomb et al. [62] were able to show that DNA from resting stages, such as spores and cysts, was only present in total DNA extractions and not in cDNA extractions from the same sample because cDNA libraries reflect only metabolically active cells.
Figure 4 shows the results of the 16S rRNA gene-based T-RFLP analysis, with the graphs representing an average of three technical replicates performed for each sample.> Genomic DNA extractions from samples of the six sampling periods P0 P5 were performed this time using the PowerSoil DNA Isolation Kit (Mobio Laboratories Inc., Carlsbad, CA, USA) in accordance with the manufacturers' protocol.
