title: Soil microbome from soil treated with carbohydates: D-Galactose
design: Soil Induced Respiration (SIR) - were measured using the MicroRespTM plate (Campbell et al. 2003). The data obtained had yield values for: (1) microbial biomass; (2) CO2 evolution without any additional substrate determined the basal respiration and with substrates addition determined substrated utilization and (3) microbial functional diversity. In order to do so fifteen different carbon sources (Table 1) of carbohydrates, carboxylic acids, amino acids and aromatic carboxylic acids was added to whole soil samples in deep well plates. CO2 evolution was measured by dye plates- a colorimetric reaction which absorbent alkali with the ability to measure carbon dioxide evolution, to determine the carbon utilization ability for each substrate. The plates with the soil and substrates added were left for one hour in order to allow acidic substrates to eliminate CaCO3-derived CO2 evolution (Oren and Steinberger, 2008). Soil sample from the same batch of soil used for SIR were used further for microbial community composition analysis.soil DNA was extracted from 0.5 g of soil sample from each sample using the GeneAll ExgeneTM mini Kit (Seoul, Korea), according to the manufacturer's instructions.  A pair of primers were used in order to express the bacterial region.  PCR amplification, using SimpliAmpTM thermal cycler (Thermo Fisher Scientific, Walham, MA, USA), by mixing 12.5 L HS Taq Mix Red (PCR Biosystems, London, UK), 9.5 L ultrapure water, 1.0 L extracted DNA, 1 l CS1-515F (ACACTGACGACATGGTTCTACAGTGCCAGCMGCCGCGGT) and 1 l CS2-806R (TACGGTAGCAGAGACTTGGTCTGGACTACHVGGGTWTCT). The thermal cycling program was set to 95C for 3 min, 24 cycles of 98C for 10 sec, 55C for 10 sec, 72C for 20 sec, and after the cycles, 72C for 1 min. The amplified DNA was stored at 20C until sequencing. All final PCR products were run on an agarose gel to verify amplification specificity and quality, in parallel with a negative control. The final PCR products were conducted by Hylabs Inc., using the Fluidigm Access Array primers for Illumina to generate libraries compatible for sequencing on the Miseq. Samples were measured for concentration by Qubit and size by Tapestation and then sequenced on the Illumina Miseq using a Miseq V2 sequencing kit (500 cycles) to generate 2x250 paired end reads.
library_name: Carbohydrates_16S_D-Galactose1