Alex007
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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 indicateselectrophesisMolecular AnalysisBefore DNA extraction, root samples were washed from CTABbuffer and then homogenized in 2-ml Eppendorf tube usingtwo 3-mm tungsten carbide beads in Mixer Mill MM400 (RetschGmbH, Haan, Germany) at 30 Hz for 5 min. The PowerSoilDNA Isolation Kit (MoBio, Carlsbad, CA, United States)was used to extract DNA from homogenized root samplesfollowing the manufacturer’s protocols. PCR was carried outusing a mixture of five forward primers ITS3ngsMixTag1-5(CTAGACTCGTCAHCGATGAAGAACGYRG) in equimolarconcentration and a degenerate reverse primer ITS4ngs(TCCTSCGCTTATTGATATGC; Tedersoo et al., 2014b). TheITS4ngs primer was supplemented with unique 10–12 basepairs long tags per sample (Supplementary Table S1). Tagswere modified from those recommended by Roche (Basel,Switzerland) to differ by >3 bases, to start only with adenosineand to comprise similar proportions of adenosine and thymidine(between 30 and 70%) to equalize their affinities in an adapterligation step (Tedersoo et al., 2014b). The PCR mixture comprised0.6 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 wascarried out in two replicates in the following thermocyclingconditions: an initial 15 min at 95°C, followed by 30 cyclesof 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, and afinal cycle of 10 min at 72°C. PCR products (typically350–400 bp) from replicate samples were pooled and theirrelative quantity was estimated by running 5 μl DNA on1% agarose gel for 25 min. DNA samples with no visiblebands were re-amplified with 35 cycles and DNA sampleswith strong bands were re-amplified with 25 cycles. Bothnegative and positive controls were included in PCR andsequencing runs. PCR products were pooled at approximatelyequimolar ratio as determined by gel band strength. Sampleswere combined into two libraries that were purified byFavorPrep™ Gel/PCR Purification Kit (Favorgen-Biotech Corp.,Austria), following the manufacturer’s instructions. DNA fromeach library was quantified using Qubit® 2.0 Fluorometer(Invitrogen, Life Technologies, CA, United States) and dsDNAHigh Sensitivity assay kit (ThermoFisher Scientific, Waltham,United States). Amplicons were pooled into two libraries andsubjected to adaptor ligation and Illumina MiSeq sequencing(2 × 300 paired-end) in NERC Biomolecular Analysis Facility(Liverpool, United Kingdom).0
Help PCR fungi
in Genetics
Posted · Edited by Alex007
thanks a lot. unfortunately, however, I cannot understand the steps following the electrophoresis. If you give me a summary I am grateful