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).
