Computational protocol: Comparison of traditional field retting and Phlebia radiata Cel 26 retting of hemp fibres for fibre-reinforced composites

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Protocol publication

[…] Hemp fibres were isolated manually from stems by removing xylem using a scalpel. Approximately 50 mg of sample (2 mm2) placed into 2 mL Eppendorf tubes was extracted directly for obtaining genomic DNA using PowerBiofilm™ DNA Isolation Kit (MO-BIO, Carlsbad, USA) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) amplification for both bacterial and fungal DNA was carried out using a C1000™ thermo-cycler (BIO-RAD, Hercules, USA). Each DNA sample (1 μL) was used as template in the PCR reactions (Sun et al. ). The universal bacterial 16S ribosomal ribonucleic acid (16S rRNA) primers used were 27F (5′-AGAGTTTGATCATGGCTCA-3′) and 1492R (5′-CGGTTACCT TGTTACGACTT-3′). The fungal primer set was ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) (Eurofins Genomics, Ebersberg, Germany) (White et al. ; Gardes et al. ). The internally transcribed spacer (ITS) is the DNA situated between the small-subunit rRNA and the large-subunit rRNA genes in the chromosome or the correspondingly transcribed region in the polycistronic rRNA precursor transcript. Extracted DNA (1 μL) was added to a PCR master mix (49 μL) containing 0.5 μM of primers, Phusion HF buffer (F-518), 200 μM dNTPs and 0.5 U Phusion Hot Start II DNA polymerase (#F-549L; Thermo Fisher Scientific, Waltham, USA) (Ale et al. ).PCR products were purified using GFX PCR DNA and Gel Band Purification Kit (GE28-9034-70 Sigma-Aldrich, Gillingham, UK). Cloning was performed using the pJet1.2/Blunt cloning vector (50 ng/µL) and T4 DNA ligase (5 U/µL). Ligation was carried out according to the manufacturer’s instructions (CloneJET PCR Cloning Kit #K1231, Thermo Scientific, USA) and the ligated product was used for transformation of electro-competent E. coli DH5α using BioRad Micropulser (BioRad, Hercules, USA). Purified plasmids were sequenced using the 27F primer for bacteria and the ITS4 primer for fungi synthesized by the company Macrogen Europe (Amsterdam, The Netherlands). The identified sequences are published in the EMBL Nucleotide Sequence Database with the accession numbers LT622055-LT622085 outlined in Tables  and for bacteria and fungi, respectively.A GenBank nucleotide database search was conducted using the BLAST algorithm (Basic Local Alignment Search Tool) to determine the closest relative of partial 16S gene sequences (Maidak et al. ). For each DNA sequence, a multiple alignment was created by Clustal W (Thompson et al. ). Evolutionary analysis and a phylogenic tree were constructed in Mega 6.0 with the Kimura two-parameter model (Kimura ). The reliability of the branches was evaluated with non-parametric bootstrapping (100 replicates). All positions with less than 95% site coverage were eliminated (complete deletion option). That means that fewer than 5% alignment gaps, missing data and ambiguous bases were allowed at any position. […]

Pipeline specifications

Software tools BLASTN, Clustal W
Application Phylogenetics
Organisms Phlebia radiata, Cannabis sativa
Diseases Fractures, Bone