Amplification of the targeted genes was carried out using gene-specific primers (S1) and DreamTaq Green PCR Master mix 2× (Thermo Fisher Scientific, cat no. K1081). The PCR contained 10 μL of DreamTaq Green PCR master mix, 0.8 μL of each template cDNA and 0.4 μL of both forward and reverse primers (10 μmol) (S1) added to a PCR tube (0.2 mL) and nuclease-free water to a final volume of 20 μL. The PCR product was electrophoresed on a 1.2% agarose gel and sequenced. The reverse compliment of the obtained nucleotide sequence of target genes was analyzed using RNAi Designing Tool (BLOCK iT™ RNAI Designer) to select the best region. The most efficient gene fragment edited to replace A from ATG (start codon) with G to prevent the start of translation. The 491 bp, 475 bp, 479 bp and 379 bp gene fragments by g Blocks® Gene Fragments, IDT (S1) of v-ATPase-A, AChE, EcR and egfp were synthesized respectively.
Dreamtaq green pcr master mix 2
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Lithuania
DreamTaq Green PCR Master Mix (2×) is a ready-to-use solution for performing polymerase chain reaction (PCR) experiments. It contains Taq DNA polymerase, dNTPs, MgCl2, and a green dye for direct gel loading.
Automatically generated - may contain errors
Lab products found in correlation
Fungus genomic dna extraction kit, by Sangon (2 mentions)
Lasergene v7, by DNASTAR (2 mentions)
Abi prism 3730 genetic analyzer, by Thermo Fisher Scientific (2 mentions)
Tprofessional standard thermocycler, by Analytik Jena (2 mentions)
Red safe, by Merck Group (1 mentions)
Phusion hi fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Restriction enzyme, by Thermo Fisher Scientific (1 mentions)
Genejet plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
Nucleospin plasmid, by Macherey-Nagel (1 mentions)
Purelink quick gel extraction kit, by Thermo Fisher Scientific (1 mentions)
Pcr2.1 topo vector, by Thermo Fisher Scientific (1 mentions)
Optipro serum free medium, by Thermo Fisher Scientific (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Prime a gene labeling system, by Promega (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Protoscript 2 first strand cdna synthesis kit, by New England Biolabs (1 mentions)
Qiaamp viral rna kit, by Qiagen (1 mentions)
Primescript 1st strand cdna synthesis kit, by Takara Bio (1 mentions)
34 protocols using dreamtaq green pcr master mix 2
1
Targeted Gene Amplification and Editing
2
Genotyping αMHC-cre Transgenic Mice
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The αMHC-cre mice were generously donated by Dr. Patrick Burgon (University of Ottawa, Faculty of Medicine, Ottawa) for our experiments. The mouse colony was maintained through backcrossing transgenic males with wild-type C57B6 females (Charles Rivers Laboratories, Wilmington, MA, USA). Mice were genotyped by extracting genomic DNA from ear clips by boiling for 10 min at 95 °C in 180 μL of 50 mM NaOH per ear. DNA solution was then neutralized using 20 μL of 1 M Tris-Cl, pH 8.0. The αMHC-cre (Cre+) or wild-type (Wt) mice were identified by polymerase chain reaction (PCR) using a DreamTaq Green PCR Master Mix 2× (Thermo Fisher Scientific, Waltham, MA, USA) for the PCR mix. To determine Cre+ animals, we utilized forward primer Cre-F (5′-ACG ACC AAG TGA CAG CAA TG-3′) and reverse primer Cre-R (5′-AAC CAG CGT TTT CGT TC-3′). PCRs were visualized by Red Safe (Sigma-Aldrich, St. Louis, MO, USA) staining on a 1% agarose gel.
Mice were handled in accordance with the guidelines set by Canadian Council on Animal Care, Guide to the Care and Use of Experimental Animals, 2 vols. (Ottawa, Ont.: CCAC, 1980-1993) and Animals for Research Act, R.S.O. 1990, c.A. 22. All animal protocols and procedures were approved by the Animal Care Committee of the University of Ottawa.
Mice were handled in accordance with the guidelines set by Canadian Council on Animal Care, Guide to the Care and Use of Experimental Animals, 2 vols. (Ottawa, Ont.: CCAC, 1980-1993) and Animals for Research Act, R.S.O. 1990, c.A. 22. All animal protocols and procedures were approved by the Animal Care Committee of the University of Ottawa.
3
Oligonucleotides and Sequencing Protocols
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Oligonucleotides used in this study are listed in Supplementary Data 2 . gBlocks designed for capturing of GPA BGCs are listed in Supplementary Table 1 . Oligonucleotides and gBlocks were ordered from Integrative DNA Technologies (Coralville, IA, USA) and Sanger and genome sequencing were performed at the MOBIX Lab Central Facility (McMaster University). PCR reactions were performed using Dream Taq Green PCR Master Mix (2×) and Phusion Hi-Fidelity DNA polymerase (Thermo Fisher Scientific). Plasmids were purified using the GeneJet Plasmid Miniprep Kit (Thermo Fisher Scientific) except for pGP1529, pGP1416, and pGP6738 which were purified using the alkaline lysis method. Restriction enzymes were purchased from Thermo Fisher Scientific and T4 DNA ligase was purchased from New England Biolabs.
4
PCR Amplification Protocol for DNA
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The final volume for the PCR cycling operation was 25 μl which included 12.5 μl of DreamTaq Green PCR Master Mix (2×) from Thermo Scientific, 1 μl of each primer at a concentration of 20 pmol, 5.5 μl of water, and 5 μl of DNA template. The carried-out reaction was in an Applied Biosystem 2720 thermal cycler under the following cycling conditions: 94°C for 5 min, followed by 35 cycles of initial denaturation at 94°C for 30 sec, primer annealing (TA) as stated in Table 1 for 40 sec, and primer extension at 72°C for 7 min.
5
Cloning and Sequencing of Duck LEAP-2
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The primers were designed using DNASTAR (DNASTAR Incorporation, Madison, WI, USA) for amplification of the LEAP-2 open reading frame from the predicted duck LEAP-2 cDNA sequence (ENSAPLT00000011688.1). The PCR product was amplified using the specific primers forward 5′-CG GGATCC ATGCACTCTTTGAAAGTCATGGC-3′ and reverse 5′-CGGAATTC CTCGGAGGCGGATCTGAG-3′ (BamHI and EcoRI restriction enzyme sites are underlined) with a DreamTaq Green PCR Master Mix (2×) (Thermo Scientific, USA). The PCR amplification was achieved under the following condition: a pre-denaturation step at 95°C for 5 min, a denaturing step at 94°C for 45 s, an annealing step at 55°C for 45 s, an extension step at 72°C for 45 s for 35 cycles, and a final extension at 72°C for 5 min. The PCR products were purified using the PureLink Quick Gel Extraction Kit (Invitrogen, USA), cloned into the pCR2.1-TOPO vector (Invitrogen, USA), and transformed using Escherichia coli (E. coli) TOP 10 competent cells (Invitrogen, USA) according to the manufacturer’s protocol. Through blue-white screening, the positive clones were picked out and then cultured overnight in Luria-Bertani (LB) broth (with 50 μg/mL ampicillin). Plasmids were extracted using NucleoSpin Plasmid (Macherey-Nagel, Düren, North Rhine-Westphalia, Germany) and sequenced at Genotech (Daejeon, Korea).
6
BLV env Gene Amplification by PCR
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The BLV env gene was amplified by PCR using specific pairs of primer that were previously evaluated by Asfaw et al. [29 (link)].
The forward primer was env gene env-F (5′-TCTGTGCCAAGTCTCCCAGATA-3′), and the reverse primer was env-R (5′-AACAACAACCTCTGGGAAGGGT-3′) to amplify the gp51 encoding region. The PCR assay was performed in 25 µL volume, comprised of 12.5 µL Dream Taq Green PCR master mix (2×) (Thermo Scientific, Schwerte, Germany), 1 µL from each primer (20 pmol/), 5.5 µL nuclease-free water, and 5 µL DNA template. Amplification was carried out by initial denaturation at 95 °C for 5 min followed by 40 amplification cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 1 min, then a final extension of 72 °C for 5 min. The PCR products were electrophoresed on 1.5% agarose gel, stained by ethidium bromide, and visualized by UV.
The forward primer was env gene env-F (5′-TCTGTGCCAAGTCTCCCAGATA-3′), and the reverse primer was env-R (5′-AACAACAACCTCTGGGAAGGGT-3′) to amplify the gp51 encoding region. The PCR assay was performed in 25 µL volume, comprised of 12.5 µL Dream Taq Green PCR master mix (2×) (Thermo Scientific, Schwerte, Germany), 1 µL from each primer (20 pmol/), 5.5 µL nuclease-free water, and 5 µL DNA template. Amplification was carried out by initial denaturation at 95 °C for 5 min followed by 40 amplification cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 1 min, then a final extension of 72 °C for 5 min. The PCR products were electrophoresed on 1.5% agarose gel, stained by ethidium bromide, and visualized by UV.
7
Sperm Cell Transfection and Fusion Assay
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On the basis of the split-GFP complementation approach (47 ), all cells were transfected using Lipofectamine 2000 (Thermo Fisher Scientific, 11668030). Sperm cells (5 × 106 cells) were transfected with 2 μg of GFP plasmid (Addgene, no. 101865) in OptiPRO serum-free medium (Thermo Fisher Scientific, 12309019) for 1 hour. Then, cells were washed with PBS three times, pelleted, resuspended in SpermPrep capacitation medium (Origio), and used for fusion reaction. Transfection efficacy of sperm cells was analyzed by PCR reaction using DNA lysis buffer [1 M tris (pH 8.5), 1 M NaCl, 0.5 M EDTA, and 10% SDS] for isolation of DNA (samples were heated for 30 min at 55°C and then for 10 min at 95°C) and DreamTaq Green PCR Master Mix 2× for PCR reaction (Thermo Fisher Scientific, K1081). HEK293T cells were transfected with FcRL3 plasmid (Origene, SC125617), IZUMO 1R (Origene, SC329497), or a combination of both. In all cases, 2 μg of plasmid was used per 5 × 105 cells in combination with Lipofectamine in Opti-MEM medium (Thermo Fisher Scientific, 31985062). List of primers used for detection of plasmid DNA after transfection is as follows: IZUMO1 , FCRL3 , and IZUMO1R (JUNO ): VP1.5, 5′-GGACTTTCCAAAATGTCG-3′ (forward); XL39, 5′-ATTAGGACAAGGCTGGTGGG-3′ (reverse): pKAM GFP : RRE, 5′-CAGGAAGCACTATGGGCGCAGC-3′ (forward); EGFPC5, 5′-CATGGTCCTGCTGGAGTTCGTG -3′ (reverse).
8
Transcriptional Profiling of Arabidopsis Tissues
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Anthers were collected from A. thaliana stage 10–12 flower buds (Smyth et al., 1990 ), stigmas from open flowers of the A. thaliana A9‐barnase line, roots from 2‐wk‐old seedlings grown on 0.5× MS plates and leaves from fully‐grown rosettes. RNA was extracted using a PureLink® RNA Mini Kit (Thermo Fisher Scientific, Loughborough, UK). cDNA synthesis was carried out using the ProtoScript® II First Strand cDNA Synthesis Kit (New England Biolabs, Hitchin, UK). DNA amplification utilized DreamTaq® Green PCR Master Mix (2×) (Thermo Fisher Scientific). Primers for DNA amplification are detailed in Table S1. RNA gel‐blot analysis was carried out as described previously (Doughty et al., 1998 ) using polyadenylated mRNA (450 ng) from leaves, stigmas, pollen and anthers derived from a range of bud sizes (whole flower buds for anthers of size < 2 mm). Labelling of gene‐specific BoPCP‐B probes (covering the coding region of the gene from aa residue 40 to the C‐terminus) was conducted using the Prime‐a‐Gene Labeling System (Promega), with modifications to the deoxynucleotidetriphosphates mix to permit double labelling with dATP and dCTP (α‐32P, 100 μCi, 6000 Ci mmol−1 each).
9
SARS-CoV-2 N Gene Detection via RT-PCR
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Total RNA was extracted from samples using a QIAamp Viral RNA Kit(QIAGEN, Germany), cDNA was synthesized using a PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara, Japan), following the manufacturer’s instructions. Primers reported by H. Tsunemitsu [34 (link)] were used: F-5’-GCCGATCAGTCCGACCAATC-3’; R-5’-AGAATGTCAGCCGGGGTAT-3’. The N gene was identified by targeting PCR activation at 94 ℃ for 4 min and 35 cycles of 94 ℃ for 1 min for denaturation, 1 min of primer annealing at 58℃ and 72 ℃ for 2 min for extension. The final extension was done at 72 ℃ for 7 min. The PCR assay was performed in a 25 µL volume, including 12.5 µL of Dream Taq green PCR master mix (2×) (Thermo Scientific, Germany), 1 µL of each primer (10 pmol/µL), 8.5 µL of deionized water, and 2 µL of DNA template.
10
Genotyping of MIR196A2 Polymorphism
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Table 1 shows detailed information about the studied SNPs (rs11614913 & rs6505162), their approved gene names, mature miRNAs, location (coordinates) on their respective chromosomes, and coded alleles, another allele, and minor allele frequencies, as well as their approved gene names. Genotyping of MIR196A2 (rs11614913) polymorphism was carried out by T‐ARMS‐PCR using a thermal cycler (Super Cycler, Model: SC300, Australia), as shown in Table 2 . Online tool Primer 1 for rs11614913 was used for primer designing (as shown in Table 2 ), PCR reactions were performed in each PCR tube in 30 μL of total volume. The reaction mixture was composed of 18 μL of DreamTaq Green PCR Master Mix (2×) (Catalog number: K1081, Thermo Fisher Scientific, MA, USA), 5 μL of nuclease‐free water, 2 μL of template DNA, 1 μL of forward outer primer, 1 μL of reverse outer primer, 1.5 μL of reverse inner primer, and 1.5 μL of forward inner primer. The PCR conditions for initial denaturation were 95°C for 5 min, followed by 35 cycles including final denaturation at 95°C for 40 s, annealing temperature at 64°C for 30 s, initial extension at 72°C for 40 s, followed by final extension at 72°C for 10 min.
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