Alex007

thanks a lot. unfortunately, however, I cannot understand the steps following the electrophoresis. If you give me a summary I am grateful

Hi, can you explain the various steps used in this article in steps? For example "and their relative quantity was estimated by running 5 μl DNA on 1% agarose gel for 25 min. "i think indicates electrophesis https://www.frontiersin.org/.../fmicb.2021.567961/full Molecular Analysis Before DNA extraction, root samples were washed from CTAB buffer and then homogenized in 2-ml Eppendorf tube using two 3-mm tungsten carbide beads in Mixer Mill MM400 (Retsch GmbH, Haan, Germany) at 30 Hz for 5 min. The PowerSoil DNA Isolation Kit (MoBio, Carlsbad, CA, United States) was used to extract DNA from homogenized root samples following the manufacturer’s protocols. PCR was carried out using a mixture of five forward primers ITS3ngsMixTag1-5 (CTAGACTCGTCAHCGATGAAGAACGYRG) in equimolar concentration and a degenerate reverse primer ITS4ngs (TCCTSCGCTTATTGATATGC; Tedersoo et al., 2014b). The ITS4ngs primer was supplemented with unique 10–12 base pairs long tags per sample (Supplementary Table S1). Tags were modified from those recommended by Roche (Basel, Switzerland) to differ by >3 bases, to start only with adenosine and to comprise similar proportions of adenosine and thymidine (between 30 and 70%) to equalize their affinities in an adapter ligation step (Tedersoo et al., 2014b). The PCR mixture comprised 0.6 μl template DNA, 0.5 μl each of the primers (20 μM), 5 μl 5 × HOT FIREPol Blend Master Mix (Solis Biodyne, Tartu, Estonia), and 13.4 μl double-distilled water. PCR was carried out in two replicates in the following thermocycling conditions: an initial 15 min at 95°C, followed by 30 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, and a final cycle of 10 min at 72°C. PCR products (typically 350–400 bp) from replicate samples were pooled and their relative quantity was estimated by running 5 μl DNA on 1% agarose gel for 25 min. DNA samples with no visible bands were re-amplified with 35 cycles and DNA samples with strong bands were re-amplified with 25 cycles. Both negative and positive controls were included in PCR and sequencing runs. PCR products were pooled at approximately equimolar ratio as determined by gel band strength. Samples were combined into two libraries that were purified by FavorPrep™ Gel/PCR Purification Kit (Favorgen-Biotech Corp., Austria), following the manufacturer’s instructions. DNA from each library was quantified using Qubit® 2.0 Fluorometer (Invitrogen, Life Technologies, CA, United States) and dsDNA High Sensitivity assay kit (ThermoFisher Scientific, Waltham, United States). Amplicons were pooled into two libraries and subjected to adaptor ligation and Illumina MiSeq sequencing (2 × 300 paired-end) in NERC Biomolecular Analysis Facility (Liverpool, United Kingdom).