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10 protocols using aligner version 3

1

Mitochondrial DNA Barcoding of Fish

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For two individual fish, total genomic DNA was isolated from absolute ethanol fixed olfactory lamellae using a QIAamp DNA Mini Kit, as per the manufacturer’s instructions. The 5′ region of the mitochondrial cytochrome c oxidase (COI) subunit I gene was amplified with the primers FishF1 (5′–TCAACCAACCACAAAGACATTGGCAC–3′) and FishR1 (5′–TAGACTTCTGGGTGGCCAAAGAATCA–3′) [39 (link)]. PCR reactions were performed in 20 μL, containing 1 ng of DNA, 1× CoralLoad PCR buffer, 3 mM MgCl2, 66 μM of each dNTP, 0.15 μM of each primer, and 0.5 units of Taq DNA polymerase (Qiagen). The amplification protocol was 4 min at 94 °C, followed by 40 cycles at 94 °C for 30 s, 48 °C for 40 s, and 72 °C for 50 s, with a final extension at 72 °C for 7 min. PCR products were purified (Ampure XP Kit, Beckman Coulter) and sequenced in both directions on a 3730xl DNA Analyzer 96-capillary sequencer (Applied Biosystems). We used CodonCode Aligner version 3.7.1 software (CodonCode Corporation, Dedham, MA, USA) to edit sequences, which were 651 bp in length, compared them to the GenBank database content with BLAST, and deposited them in GenBank under accession numbers MN397913 and MN397914. Species identification was confirmed with the BOLD identification engine [41 ].
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2

Mitochondrial DNA Barcoding of Fish

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Total genomic DNA was isolated using a QIAamp DNA Mini Kit (Qiagen, Courtaboeuf, France), as per the manufacturer’s instructions. The 5′ region of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified with the primers FishF1 (5′ – TCAACCAACCACAAAGACATTGGCAC – 3′) and FishR1 (5′ – TAGACTTCTGGGTGGCCAAAGAATCA – 3′) [75 (link)]. PCR reactions were performed in 20 μL, containing 1 ng of DNA, 1× CoralLoad PCR buffer, 3 mM MgCl2, 66 μM of each dNTP, 0.15 μM of each primer, and 0.5 units of Taq DNA polymerase (Qiagen). The amplification protocol was 4 min at 94 °C, followed by 40 cycles at 94 °C for 30 s, 48 °C for 40 s, and 72 °C for 50 s, with a final extension at 72 °C for 7 min. PCR products were purified (Ampure XP Kit, Beckman Coulter) and sequenced in both directions on a 3730 × l DNA Analyzer 96-capillary sequencer (Applied Biosystems, Foster City, CA, USA). We used CodonCode Aligner version 3.7.1 software (Codon Code Corporation, Dedham, MA, USA) to edit sequences, compared them to the GenBank database content with BLAST, and deposited them in GenBank under accession numbers MT666082MT666086. Species identification was confirmed with the BOLD identification engine [68 (link)].
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3

Mitochondrial DNA Sequencing of Fish

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Total genomic DNA was isolated using a QIAamp DNA Mini Kit (Qiagen), as per the manufacturer’s instructions. The 5′ region of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified with the primers FishF1 and FishR1 (Ward et al., 2005 (link)). PCR reactions and amplification were performed as in Ayadi et al. (2017) (link). We used CodonCode Aligner version 3.7.1 software (CodonCode Corporation, Dedham, MA, USA) to edit sequences, which were 655 bp in length, compared them to the GenBank database content with BLAST, and deposited them in GenBank under accession numbers MG761757MG761759 and MW788679MW788687 (Table 1). Species identification was confirmed with the BOLD identification engine (Ratnasingham & Hebert, 2007 (link)).
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4

Mitochondrial DNA Barcoding Protocol

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Total genomic DNA was isolated using a QIAamp DNA Mini Kit (Qiagen, Courtaboeuf, France), according to the manufacturer’s instructions. The 5′ region of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified with the primers TelF1 (5′–TCGACTAATCAYAAAGAYATYGGCAC–3′) and TelR1 (5′–ACTTCTGGGTGNCCAAARAATCARAA–3′) [21 ]. PCR reactions were performed in 20 μL, containing 1 ng of DNA, 1× CoralLoad PCR buffer, 3 mM MgCl2, 66 μM of each dNTP, 0.15 μM of each primer, and 0.5 units of Taq DNA polymerase (Qiagen). The amplification protocol was 4 min at 94 °C, followed by 40 cycles at 94 °C for 30 s, 48 °C for 40 s, and 72 °C for 50 s, with a final extension at 72 °C for 7 min. PCR products were purified (Ampure XP Kit, Beckman Coulter, Brea, CA, USA) and sequenced in both directions on a 3730 × l DNA Analyzer 96-capillary sequencer (Applied Biosystems, Foster City, CA, USA). We used CodonCode Aligner version 3.7.1 software (Codon Code Corporation, Dedham, MA, USA) to edit sequences, compared them to the GenBank database content with BLAST, and deposited them in GenBank (accession numbers in Table 1). Species identification was confirmed with the BOLD identification engine [67 (link)].
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5

Mitochondrial COI Gene Sequencing Protocol

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Total genomic DNA was isolated using QIAamp DNA Mini Kit (Qiagen, Courtaboeuf, France) as per the manufacturer's instructions. The 5′ region of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified with the primers TelF1 (5′-TCGACTAATCAYAAAGAYATYGGCAC-3′) and TelR1 (5′-ACTTCTGGGTGNCCAAARAATCARAA-3′) (Dettaï et al. 2011) . PCR reactions were performed in 20 μl, containing 1 ng of DNA, 1× CoralLoad PCR buffer, 3 mM MgCl2, 66 μM of each dNTP, 0.15 μM of each primer, and 0.5 units of Taq DNA polymerase (Qiagen). The amplification protocol was 4 min at 94 °C, followed by 40 cycles at 94 °C for 30 s, 48 °C for 40 sec, and 72 °C for 50 s, with a final extension at 72 °C for 7 min. PCR products were purified (Ampure XP Kit, Beckman Coulter) and sequenced in both directions on a 3730xl DNA Analyzer 96-capillary sequencer (Applied Biosystems, Foster City, USA). We used the CodonCode Aligner version 3.7.1 software (Codon Code Corporation, Dedham, MA, USA) to edit sequences, compared them to the GenBank database content with BLAST, and deposited them in GenBank under accession numbers MW484937-MW484939. Species identification was confirmed with the BOLD identification engine (Ratnasingham and Hebert 2007) (link).
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6

Mitochondrial DNA Barcoding for Species ID

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Total genomic DNA was isolated using QIAamp DNA Mini Kit (Qiagen, Courtaboeuf, France)
as per the manufacturer's instructions. The 5 ʹ region of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was amplified with the primers FishF1 (5 ʹ -TCA ACC AAC CAC AAA GAC ATT GGC AC-3 ʹ ) and FishR1 (5 ʹ -TAG ACT TCT GGG TGG CCA AAG AAT CA-3 ʹ ) (Ward et al., 2005) . PCR reactions were performed in a total volume of 20 µl, containing 1 ng of DNA, 1× CoralLoad PCR buffer, 3 mM MgCl 2 , 66 µM of each dNTP, 0.15 µM of each primer and 0.5 units of Taq DNA polymerase (Qiagen). The amplification protocol was 4 min at 94°C, followed by 40 cycles at 94°C for 30 s, 48°C for 40 s, and 72°C for 50 s, with a final extension step at 72°C for 7 min. PCR products were purified (Ampure XP Kit, Beckman Coulter, Brea, USA) and sequenced in both directions on a 3730xl DNA Analyzer 96-capillary sequencer (Applied Biosystems, Foster City, USA). We used CodonCode Aligner version 3.7.1 software (CodonCode Corporation, Dedham, MA, USA) to edit the sequence, which was 652 bp in length, compared it to the GenBank database content with BLAST, and deposited it in GenBank under the accession number MK275650. Species identification was confirmed with the BOLD identification engine (Ratnasingham & Hebert, 2007) .
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7

DNA Barcoding of Fish Species

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We used the QIAamp DNA Mini Kit (Qiagen), per the manufacturer’s instructions, to perform DNA extraction. The 5′ region of the cytochrome oxidase I (COI) mitochondrial gene was amplified with the primers FishF1 (5′-TCAACCAACCACAAAGACATTGGCAC-3′) and FishR1 (5′-TAGACTTCTGGGTGGCCAAAGAATCA-3′) (Ward et al., 2005 (link)). PCR reactions were performed in 20 µl, containing 1 ng of DNA, 1x CoralLoad PCR buffer, 3 mM MgCl2, 66 µM of each dNTP, 0.15 µM of each primer, and 0.5 units of Taq DNA polymerase (Qiagen). The amplification protocol was 4 min at 94 °C, followed by 40 cycles at 94 °C for 30 s, 48 °C for 40 s, and 72 °C for 50 s, with a final extension at 72 °C for 7 min. PCR products were purified (Ampure XP Kit; Beckman Coulter) and sequenced in both directions on a 3730xl DNA Analyzer 96-capillary sequencer (Applied Biosystems). We used CodonCode Aligner version 3.7.1 software (CodonCode Corporation, Dedham, MA, USA) to edit sequences, which were 670 bp in length, compared them to the GenBank database content with BLAST, and deposited them in GenBank under accession numbers KT023566, KT023567, KT023568 and KU739501KU739517. Species identification was confirmed with the BOLD identification engine (Ratnasingham & Hebert, 2007 (link)).
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8

Metabarcoding of Fish Mitochondrial COI Gene

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Total genomic DNA was isolated using QIAamp DNA Mini Kit (Qiagen) as per the manufacturer's instructions. The 5′ region of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified with the primers FishF1 (5′-TCAACCAACCACAAAGACATTGGCAC-3′) and FishR1 (5′-TAGACTTCTGGGTGGCCAAAGAATCA-3′) [19] . PCR reactions were performed in 20 μl, containing 1 ng of DNA, 1× CoralLoad PCR buffer, 3 mM MgCl2, 66 μM of each dNTP, 0.15 μM of each primer, and 0.5 units of Taq DNA polymerase (Qiagen). The amplification protocol was 4 min at 94°C, followed by 40 cycles at 94°C for 30 sec, 48°C for 40 sec, and 72°C for 50 sec, with a final extension at 72°C for 7 min.
PCR products were purified (Ampure XP Kit, Beckman Coulter) and sequenced in both directions on a 3730xl DNA Analyzer 96-capillary sequencer (Applied Biosystems). We used CodonCode Aligner version 3.7.1 software (CodonCode Corporation, Dedham, MA, USA) to edit sequences, which were 652 bp in length, compared them to the GenBank database content with BLAST, and deposited them in GenBank under accession numbers KX926437 -KX926442. Species identification was confirmed with the BOLD identification engine [20] .
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9

Molecular Profiling of Microbial Communities

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All bands in the DGGE gel of 16S rRNA fragments were excised with a razor blade and soaked in DNA-free water at 4°C overnight. The DNA sample was then subjected to PCR amplification using the EUB f933 and EUB r1387 primers (11 (link)). PCR products were then purified with the MonoFas PCR Purification Kit and cloned into the pGEM-T-easy Vector System (Promega) using Escherichia coli JM109 as the host. The clones were then subjected to PCR using the M13 primers. The diluted PCR products were then further amplified using the GC-clamp-EUB f933 and EUB r1387 primers, after which DGGE was performed. Clones were sequenced and then phylogenetically analyzed. The sequencing of selected clones was performed on a CEQ8000 DNA Genetic Analysis System (Beckman Coulter, Indianapolis, IN, USA) with M13 primers. Base-calling of sequence trace data was performed using the CodonCode Aligner version 3.7.1 (CodonCode Corporation, Dedham, MA, USA). Sequences were aligned using Clustal W, and phylogenetic trees were constructed using the Maximum-likelihood method. Sequence homologies were searched with the blastn program from the DNA Data Bank of Japan (DDBJ; http://www.ddbj.nig.ac.jp).
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10

HLA-G Allele Typing by PCR and Sequencing

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Allele typing was carried out by PCR and direct sequencing from all HBEC cDNA using JEG-3 cDNA as control (G*01:01; G*01:01, exon8 14 bp ins/ins) (18 (link)). PCR amplification was performed using a Multiplex PCR Kit (Qiagen) following manufacturer’s recommendations with primers targeting HLA-G exon 1 to exon 5 (Table S1 in Supplementary Material). Direct sequencing was performed using Big Dye Terminator V1.1 kit (Life Technologies) according to the manufacturer’s protocol. Alignments were performed with Codon Code Aligner version 3.7.1 (Codon Code Corporation, USA) using HLA-G allele sequences from the official IMGT/HLA database 3.20.0 May 2015 (36 (link)).
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