The pair of universal ITS primers ITS-1 (forward) and ITS-4 (reverse) was used for the Sanger sequencing of the purified PCR products using model ABI PRISM® 3500XL DNA Sequencer (Applied Biosystems, Foster City, CA, USA) following manufacturer’s instructions. For good quality sequence assurance, FinchTV version1.4.0 software was used for the analysis of sequences (sense and antisense) resulting from sequencing reaction (Geospiza, Inc.; Seattle, WA, USA; https://digitalworldbiology.com/FinchTV , http://www.geospiza.com (accessed on 5 August 2021)). The resulting sequences were edited using BioEdit Sequence Alignment Editor (Ibis Therapeutics, Carlsbad, CA, USA). Then, the resulting consensus ITS sequences were blasted in the NCBI (http://www.ncbi.nlm.nih.gov , accessed on 5 August 2021) database with the BLASTn for homology in order to identify the probable mushroom in question. The sequences were deposited in the GenBank®.
Abi prism 3500xl dna sequencer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI PRISM® 3500XL DNA Sequencer is a capillary electrophoresis-based instrument designed for DNA sequencing applications. It utilizes four-color fluorescence detection technology to analyze DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Prism ready reaction dye terminator cycle sequencing kit, by Thermo Fisher Scientific (2 mentions)
C1000 thermal cycler, by Bio-Rad (1 mentions)
Dna spin plasmid dna purification kit, by iNtRON Biotechnology (1 mentions)
Dreamtaq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Zr fungal bacterial dna kit, by Zymo Research (1 mentions)
7 protocols using abi prism 3500xl dna sequencer
1
ITS Sequencing for Mushroom Identification
2
16S rRNA Gene Sequencing from Soil
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The extraction of genomic DNA was carried out using the ZR soil microbe DNA (Zymo Research) kit as described in the kit protocol. The 16S rRNA gene nucleotide sequences were determined by PCR using a C1000 thermal cycler (BioRad, CA, USA). The fD1 (5′-AGAGTTTGATCCTGGCTCAG-3′) and rP2 (5′-ACGGCTACCTTGTTACGACTT-3′) primers were used for amplification [19 (link)]. A final volume of 25 μl was used for the PCR reactions, which contained 12.5 μl of 2× master mix (One Taq® Hot Start Quick Load, Biolabs, England), 0.5 μl of each primer, and 1 μl DNA. The thermal cycling settings were initial denaturation for 5 min at 94°C, followed by 30 cycles of 30 s at 94°C, annealing for 1 min at 54°C, and extension for 2 min at 72°C. The PCR result was sequenced using an ABI PRISM® 3500XL DNA Sequencer (Applied Biosystems) at Inqaba Biotechnical Industrial (Pty) Ltd in Pretoria, South Africa. The sequences were analyzed and edited using Bio Edit [20 ]. Evolutionary distance matrices were generated according to Jukes and Cantor [21 ]. Phylogenetic and molecular evolutionary analyses were performed with MEGA version 5.2.2 [22 (link)], and a phylogenetic tree was constructed using the neighbor-joining method [23 (link)]. Bootstrap analysis was performed using 1000 replications for the neighbor-joining data set.
3
Plasmid Isolation and Transformation
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The plasmids were isolated from the positive transformed colony of E. coli DH5α using the DNA-spin Plasmid DNA Purification Kit (iNtRON Biotechnology Inc.), and the recombinant pET 29a(+) plasmids were transformed into expression host E. coli BL21 (DE3) pLysS (Novagen) using the calcium chloride transformation protocol mentioned previously with DH5α. In the same manner, the positive transformed colony was screened by colony PCR technique as above. The sequencing was done on both the DNA strands by universal pET29a(+) forward and reverse primers using ABI PRISM 3500XL DNA Sequencer (Applied Biosystems, Waltham, MA, USA).
4
DNA Sequencing and Bioinformatic Analysis
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Amplified PCR products were sequenced using an ABI PRISM® 3500XL DNA Sequencer (Applied Biosystems) at Inqaba Biotechnical Industrial (Pty) Ltd. (Pretoria, South Africa). The acquired sequences were aligned on GenBank database using Basic Local Alignment Search Tool (BLAST) (www.ncbi.nlm.nih.gov/BLAST ) from the National Center for Biotechnology Information (NCBI) to identify sequences with high similarity.
5
Sanger Sequencing of Purified PCR Products
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The sanger sequencing of the purified PCR products was done at Inqaba Biotechnical Industrial (Pty) Ltd, Pretoria, South Africa with PRISM™ Ready Reaction Dye Terminator Cycle sequencing kit using the dideoxy chain termination method and electrophoresed with a model ABI PRISM® 3500XL DNA Sequencer (Applied Biosystems, Foster City, CA, USA) following manufacturer’s instructions.
For good quality sequence assurance, ChromasLite version 2.33 software was used for the analysis of chromatograms (sense and antisense) resulting from sequencing reaction (Technelysium 2004 ). The resulting chromatograms were edited using BioEdit Sequence Alignment Editor (Hall 2004 ). After this, the resulting consensus 16S rDNA sequences obtained were blasted in the NCBI (http://www.ncbi.nlm.nih.gov ) database with the BLASTn for homology in order to identify the probable organism in question (Altshul et al. 1997 (link)). The sequences were deposited in the GenBank.
For good quality sequence assurance, ChromasLite version 2.33 software was used for the analysis of chromatograms (sense and antisense) resulting from sequencing reaction (Technelysium 2004 ). The resulting chromatograms were edited using BioEdit Sequence Alignment Editor (Hall 2004 ). After this, the resulting consensus 16S rDNA sequences obtained were blasted in the NCBI (
6
Sequencing and Phylogenetic Analysis of 16S rRNA Genes
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Purified amplicons were sequenced at Inqaba Biotechnical Industries (Pty) Ltd., Pretoria, South Africa. The PRISM™ Ready Reaction Dye Terminator Cycle Sequencing Kit (Sanger sequencing) with reverse primer 907R (5′-CCGTCAATTCCTTTGAGTTT-3′) was employed in the sequencing PCR reactions; thereafter, the products were electrophoresed with a model ABI PRISM® 3500XL DNA Sequencer (Applied Biosystems, Foster City, CA, USA). The high-quality partial 16S rRNA gene sequences obtained from the isolates were clustered into operational taxonomic units (OTUs) at 99% similarity using the Mothur software version 1.37.2 [27 (link)]. Thereafter, taxonomies were assigned to OTU representative sequences by aligning the sequences against the EzBiocloud database (http://www.ezbiocloud.net/ accessed on 12 December 2020) [28 (link)]. MEGA version X [29 (link)] was used to phylogenetically reconstruct the OTU representative sequences with closely related GenBank sequences as previously described by Adedeji et al. [30 (link)]. All the sequences obtained in this study were deposited at NCBI GenBank under the accession numbers MW448721, MW448743, MW449471–MW449476, MW465227–MW465230, and MW473709–MW473713.
7
Bacterial 16S rRNA Sequence Analysis
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Extraction of total DNA was achieved using the ZR Fungal/Bacterial DNA Kit™ (Zymo Research, Irvine, CA, USA) according to the manufacturer's instructions. Target 16S rRNA regions were amplified using DreamTaq™ DNA polymerase (Thermo Scientific™, Thermo Fisher Scientific, Waltham, MA, USA) with the bacterial universal primers 27F (5 -AGAGTTTGATCMTGGCTCAG-3 ) and 1492R (5 -CGGTTACCTTGTTACGACTT-3 ) as described by Lane et al. [42] and Turner et al. [43] .
Sanger sequencing of the purified PCR products was carried out by Inqaba Biotechnical Industries (Pty) Ltd., Hatfield, South Africa using the PRISM™ Ready Reaction Dye Terminator Cycle sequencing kit using chain termination by the dideoxy-nucleotides methodology and electrophoresed on an ABI PRISM ® 3500XL DNA Sequencer (Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions. Resultant chromatograms were analysed (Chromas version 2.6.6) as outlined in Technelysium [44] , followed by pairwise alignment in BioEdit Sequence Alignment Editor, version 7.0.5.3 [45] , and the resultant gene sequences compared with those already deposited (https://www.ncbi.nlm.nih.gov/).
Sanger sequencing of the purified PCR products was carried out by Inqaba Biotechnical Industries (Pty) Ltd., Hatfield, South Africa using the PRISM™ Ready Reaction Dye Terminator Cycle sequencing kit using chain termination by the dideoxy-nucleotides methodology and electrophoresed on an ABI PRISM ® 3500XL DNA Sequencer (Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions. Resultant chromatograms were analysed (Chromas version 2.6.6) as outlined in Technelysium [44] , followed by pairwise alignment in BioEdit Sequence Alignment Editor, version 7.0.5.3 [45] , and the resultant gene sequences compared with those already deposited (https://www.ncbi.nlm.nih.gov/).
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