ISO 17601:2016 pdf free

ISO 17601:2016 pdf free.Soil quality一Estimation of abundance of selected microbial gene sequences by quantitative PCR from DNA directly extracted from soil
qPCR assay is based on the quantification of the amplicons at the end of each PCR cycle by using a DNA dye which fluoresces when intercalated in the double strand amplicons. The purpose of this task is to describe the definition of appropriate amplicon to settle down a qPCR assay (step one), the preparation of qPCR standard (step two), and the calibration of the qPCR assay (step three).
The first step aims at designing oligonucleotide primer pair; it can be designed in silico using different programs using the sequence of the microbial gene of interest to be quantified by qPCR from soil DNA extracts. The specificity of the primers shall be checked in silico by comparing their sequences to known sequences available in the Genbank database (http://www.ncbi.nlm.nih.gov/genbank/).
Only primers specific for the gene target shall be considered. The main parameters to be considered to design oligonucleotide primer pair for establishing qPCR assay are listed thereafter.
Step 2 of task 1 describes the procedure used to generate qPCR standards targeting a sequence of the microbial gene of interest from different DNA templates (pure bacterial or fungal isolate, environmental DNA, or artificial DNA). It also reports the procedure used to insert the qPCR standard in a cloning vector, transform Escherichia coli, and purify recombinant plasmids harboring qPCR standard for further use for qPCR assays.
Competent E. coli are transformed by heat shock as described below. Competent cells (108 cfu*μg-1 of DNA) freshly thawed out are incubated for 5 min on ice. Then 1 ul of the ligation reaction is added to cells, smoothly mixed, and incubated for 20 min on ice. Bacterial cells are heat shock treated for 50 s incubation at 42 °C and immediately placed on ice and incubated for 2 min. Then 950 μl of SOC medium are added and the bacterial cells are incubated at 37 °C under agitation at 150 min-1 for 1 h. 100 μl bacterial cells aliquots are plated onto LB/Amp/IPTG/X-Gal solid medium. Petri dishes are then incubated at 37 °C overnight.ISO 17601 pdf free download.