DNA was extracted from subgingival plaque samples by the “modified proteinase K” method as described previously [21 (link)]. A real time polymerase chain reaction (RT-PCR) was carried out for amplification of the 16S rRNA species-specific gene of P. gingivalis in our previous study [16 (link)]. Samples found to be positive for P. gingivalis were further processed for the detection of fimA genotypes I to V by polymerase chain reaction (PCR), as described previously [22 (link),23 (link)]. The primers specific to each of the fimA types used in the study were as mentioned in Table 1 . A reaction mixture with a total volume of 25 µL was prepared by using Ampliqon red 2X mastermix (Ampliqon, Odense, Denmark), which contains Tris-HCL pH 8.5, (NH4)2SO4, 3 mM MgCl2, 0.2% Tween 20, 0.4 mM of each dNTP, 0.2 units/µL Ampliqon Taq DNA polymerase, and inert red dye and stabilizer. The primers were used at 0.5 µM concentration, and 3 µL of the DNA template at approximately 100ng concentration was added to the reaction mixture. The thermal cycling conditions were performed in a veriti 96-well thermal cycler (Applied Biosystems, California, CA, USA). An initial denaturation was done at 95 °C for 5 min, followed by 35 cycles of 95 °C, 58 °C and 72 °C for 30 s each. The final extension was carried out at 72 °C for 5 min.
Taq dna polymerase
Manufactured by Ampliqon
Sourced in Denmark
Taq DNA polymerase is a thermostable enzyme isolated from the thermophilic bacterium Thermus aquaticus. It is a key component in the Polymerase Chain Reaction (PCR) process, responsible for the enzymatic replication of DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Midori green, by Nippon Genetics (5 mentions)
Master mix red, by Ampliqon (2 mentions)
Led light, by Nippon Genetics (2 mentions)
Dntps, by Thermo Fisher Scientific (2 mentions)
Veriti 96 well thermal cycler, by Thermo Fisher Scientific (1 mentions)
Dna extraction kit, by CinnaGen (1 mentions)
Generuler 1 kb plus dna ladder, by Thermo Fisher Scientific (1 mentions)
Hyperscript reverse transcriptase, by GeneAll (1 mentions)
Pyromark gold q96 reagents kit, by Qiagen (1 mentions)
Csl mdna 100bp, by Cleaver Scientific (1 mentions)
Agarose gel, by Merck Group (1 mentions)
100 bp dna ladder, by Thermo Fisher Scientific (1 mentions)
Abi 3730xl dna analyzer, by Thermo Fisher Scientific (1 mentions)
High pure pcr template preparation kit, by Roche (1 mentions)
Uv transilluminator, by Vilber (1 mentions)
Geneamp 9700 thermocycler, by Thermo Fisher Scientific (1 mentions)
Mgcl2, by Ampliqon (1 mentions)
23 protocols using taq dna polymerase
1
Identification of P. gingivalis fimA Genotypes
2
Genomic DNA Extraction and PCR Amplification
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For all PCR experiments, genomic DNA was extracted using an appropriate DNA extraction kit (Cinnagen, Karaj, Iran), according to the manufacturer’s instruction. The absorption of the extracted DNA was measured at 260 nm and 280 nm, to confirm the quality of the product.
PCR amplification was conducted in a temperature-gradient thermal cycler (Biometra-T300, Gottingen, Germany), with a total volume of 50 µl. Each 50-µl PCR mixture consisted of 2 µl of bacterial DNA, 0.2 µM of each specific primer, and 25 µl of 2× Master Mix Red (Ampliqon, Odensem, Denmark); the master mix consisted of 0.2 unit/µl of AmpliqonTaq DNA polymerase, 1.5 mM MgCl2, and 0.4 µM dNTPs. After amplification, 5 µl of the PCR product was electrophoresed on a 1.5% agarose gel in 0.5× TBE buffer (5.4 g Tris base, 2.75 g boric acid, and 2 ml 0.5-M EDTA, in 1 L). The DNA ladder was a ready-to-use plasmid double digest of 100-3000 bp, which was obtained from SMOBIO Technology (Hsinchu, Taiwan). The specificity of the primers was verified using the Primer Quest software tool (http://www.ncbi.nlm.nih.gov/Gene ).
PCR amplification was conducted in a temperature-gradient thermal cycler (Biometra-T300, Gottingen, Germany), with a total volume of 50 µl. Each 50-µl PCR mixture consisted of 2 µl of bacterial DNA, 0.2 µM of each specific primer, and 25 µl of 2× Master Mix Red (Ampliqon, Odensem, Denmark); the master mix consisted of 0.2 unit/µl of AmpliqonTaq DNA polymerase, 1.5 mM MgCl2, and 0.4 µM dNTPs. After amplification, 5 µl of the PCR product was electrophoresed on a 1.5% agarose gel in 0.5× TBE buffer (5.4 g Tris base, 2.75 g boric acid, and 2 ml 0.5-M EDTA, in 1 L). The DNA ladder was a ready-to-use plasmid double digest of 100-3000 bp, which was obtained from SMOBIO Technology (Hsinchu, Taiwan). The specificity of the primers was verified using the Primer Quest software tool (
3
Genomic DNA Extraction and SSR Marker Analysis
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Total DNA was extracted from trifoliate leaves of each of 150 H-e3 × ZE and 156 H-e3 × YU F2 plants as described by Doyle and Doyle (1990) , and from each of 492 seeds from two F2 plants from the H-e3 × ZE cross, as described by Xia et al. (2012) (link). Each PCR mixture for SSR marker analysis contained 30 ng of total genomic DNA as a template, 0.2 μl of each primer (10 μM), 0.8 μl of dNTPs (2.5 mM), 0.1 μl of Taq DNA polymerase (Ampliqon), and 1 μl of 10× ammonium buffer (Ampliqon) in a total volume of 10 μl; amplification conditions were 35 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. PCR products were separated by electrophoresis in 10.5% (w/v) polyacrylamide gels, stained with ethidium bromide, and visualized under UV light.
4
Amplification and Sequencing of Eg14-3-3 Gene
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Eg14-3-3 gene was amplified by polymerase chain reaction (PCR). The PCR reaction performed in a 25 µL reaction volume as follows: 9.5 µL DNase free deionized water, 1 µL template DNA (1017.6 ng/µL), 1 µL each primer (100 pmoles/µL) and 12.5 µL PCR master mix (2x Master Mix RED, Taq DNA polymerase, 0.5 µM of dNTPs and 1.5 mM MgCl2) obtained from Ampliqon (Odense, Denmark). Based on NCBI’s Eg14-3-3 gene sequence (GenBank accession No: AY942149), the forward and reverse primers were designed using OLIGO 7 software.16 (link) The primers were as follows: the forward primer, 5'-ATGTCTTCTCTCAGTAAGCGCGA-3' and the reverse primer, 5'-ATCGGCTTTCGGCGGTTCAG-3'. The gene amplification was conducted using following condition: (i) one cycle at 94°C for 4 minutes as primary denaturation, (ii) 32 cycles at 94 °C for 1 minute (denaturation), 52°C for 1 minute (annealing) and 72°Cfor 1 minute (extension), and (iii) three cycles as final extension (72°C for 10 minutes). The PCR products were visualized by electrophoresis on a 1% agarose gel and size of the product analyzed with 1 kb DNA ladder (GeneRulerTM 1 kb Plus DNA ladder), obtained from Fermentase (Thermo Fisher Scientific, Waltham, USA). The PCR-amplified product was sequenced, and its mRNA sequence was registered in NCBI GenBank as Iran Eg14-3-3 isolate (GenBank: KU739136).
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Eg14-3-3 gene was amplified by polymerase chain reaction (PCR). The PCR reaction performed in a 25 µL reaction volume as follows: 9.5 µL DNase free deionized water, 1 µL template DNA (1017.6 ng/µL), 1 µL each primer (100 pmoles/µL) and 12.5 µL PCR master mix (2x Master Mix RED, Taq DNA polymerase, 0.5 µM of dNTPs and 1.5 mM MgCl2) obtained from Ampliqon (Odense, Denmark). Based on NCBI’s Eg14-3-3 gene sequence (GenBank accession No: AY942149), the forward and reverse primers were designed using OLIGO 7 software.16 (link) The primers were as follows: the forward primer, 5'-ATGTCTTCTCTCAGTAAGCGCGA-3' and the reverse primer, 5'-ATCGGCTTTCGGCGGTTCAG-3'. The gene amplification was conducted using following condition: (i) one cycle at 94°C for 4 minutes as primary denaturation, (ii) 32 cycles at 94 °C for 1 minute (denaturation), 52°C for 1 minute (annealing) and 72°Cfor 1 minute (extension), and (iii) three cycles as final extension (72°C for 10 minutes). The PCR products were visualized by electrophoresis on a 1% agarose gel and size of the product analyzed with 1 kb DNA ladder (GeneRulerTM 1 kb Plus DNA ladder), obtained from Fermentase (Thermo Fisher Scientific, Waltham, USA). The PCR-amplified product was sequenced, and its mRNA sequence was registered in NCBI GenBank as Iran Eg14-3-3 isolate (GenBank: KU739136).
5
Reverse Transcription and PCR Amplification
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Total RNA of samples was isolated by Total RNA isolation kit (DENA Zist Asia). The cDNA(s) were synthesized using Hyper script reverse transcriptase (Gene All) and oligod (T) 18mer, P.SOS.S-REV1, P.SOS.S-REV2, P.SOS.S-REV3, P.SOS.S-REV4, P.NHX.S.REV1, P.NHX.S.REV2 and P.NHX.S.REV3primers (Table 1 ) and amplified with a combination of primers (Table 2 ). The amplifications were obtained in 30 cycles at defined annealing temperature for each pair of primers using TaqDNA polymerase (AMPLIQON). The process finished after a final extension for 5–15 min at 720C (Fig. 1 ).![]()
Regulation of ion homeostasis by ion Na+/H+ pumps antiporters (SOS1), vacuolar Na+/H+ exchanger (NHX) that salt sensors present at the plasma and vacuolar membranes [32 (link)]
6
Evaluating antifungal resistance in Aspergillus
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Conventional polymerase chain reaction (PCR) assay was carried out to determine the possible presence of the
resistance-related mutations in the CYP51A gene of induced resistant A. fumigatus isolate(s) (MIC>2 µg/ml)
in a total volume of 25 ml, containing 12.5 ml Taq 2×Master Mix Red 0.1 M Tris/HCl, pH 8.5, (NH4)2SO4, 4 mM MgCl2,
0.2% Tween 20, 0.4 mM deoxynucleotides, 0.2 units Taq DNA Polymerase (manufactured by Ampliqon, Denmark),
10 pmol of each primer (Table 1 ), 2 ml template DNA, and 8.5 ml distilled water.
Primers were designed to cover the promoter and full coding gene sequences. The PCR amplification started with an initial
denaturation at 95 ⁰C for one min, followed by 35 cycles of denaturation at 94 ⁰C (60 sec), 60 ⁰C (30 sec), 72 ⁰C (60 sec),
and a final 10min extension at 72 ⁰C. It must be noted that the PCR products were run on a 2% agarose gel.
resistance-related mutations in the CYP51A gene of induced resistant A. fumigatus isolate(s) (MIC>2 µg/ml)
in a total volume of 25 ml, containing 12.5 ml Taq 2×Master Mix Red 0.1 M Tris/HCl, pH 8.5, (NH4)2SO4, 4 mM MgCl2,
0.2% Tween 20, 0.4 mM deoxynucleotides, 0.2 units Taq DNA Polymerase (manufactured by Ampliqon, Denmark),
10 pmol of each primer (
Primers were designed to cover the promoter and full coding gene sequences. The PCR amplification started with an initial
denaturation at 95 ⁰C for one min, followed by 35 cycles of denaturation at 94 ⁰C (60 sec), 60 ⁰C (30 sec), 72 ⁰C (60 sec),
and a final 10min extension at 72 ⁰C. It must be noted that the PCR products were run on a 2% agarose gel.
7
Three-Primer Genotyping of Mastl Locus
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A three-primer strategy was utilized for genotyping of Mastl locus, to distinguish FLOX and KO alleles (Figure 2A). The primers used for genotyping; are given in Table S2. For the PCR genotyping, the samples were prepared according to the adapted HotSHOT protocol (Truett et al., 2000) (link). For the genotyping PCR, 0.5 µL of DNA sample and 0.25 units of Taq DNA polymerase (A111103; Ampliqon) was used for each 10 µL of reaction volume. Final primer concentrations were 1 µM for FOR and REV1, and 0.15 µM for REV2 primers. 35 PCR cycles were performed using 67 o C annealing temperature. Under these optimized conditions, the PCR reaction consistently yields roughly equal intensity bands for Mastl FLOX (300 bp) and
Mastl KO (500 bp) alleles from a heterozygous Mastl FLOX/KO cell clone (Figure 2).
Mastl KO (500 bp) alleles from a heterozygous Mastl FLOX/KO cell clone (Figure 2).
8
Pyrosequencing Protocol for SNP Analysis
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Pyrosequencing (Ronaghi et al., 1996 (link)) was performed using pyromark gold Q96 reagents kit (Qiagen) and a PSQTM 96 MA pyrosequencing instrument (Biotage AB) according to manufacturer's protocols. Gene specific cDNA or genomic DNA fragments between 100 and 300 base pairs containing the SNP of interest were amplified by PCR using the primers listed in Supplemental File S2 . The third sequencing primer was used to sequence a short region around the SNP of interest (Supplemental File S2 ). The PCR reaction was carried out in 25 μl total volume containing 2.5 μl 10x buffer (100 mM Tris-HCL pH 8.3, 500 mM KCl, 15 mM MgCl2, 1% Triton X-100), 0.2 mM dNTP Mix, 0.4 μM each forward and reverse primers, 0.5 U Taq DNA Polymerase (Ampliqon), high purity water (Merck KGaA, Darmstadt, Germany) and 50 ng template DNA. The PCR reaction was run with 2 min initial denaturation at 94°C, followed by 50 cycles with 45 s denaturation at 94°C, 45 s annealing at the temperature specified for each primer pair (Supplemental File S2 ), and extension for 1 min at 72°C. At the end of the cycles, the reaction was run for 10 min at 72°C. Reactions were generally performed in a Labcycler (SensoQuest GmbH, Göttingen).
9
Mitochondrial and Nuclear DNA Extraction and Amplification
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A standard salt extraction method (Bruford et al. 1992 ) was used to isolate DNA from the tissue sample using ATL lysis and AE elution buffers. Standard PCR procedures were utilised to amplify one partial mitochondrial ribosomal gene (ribosomal ribonucleic acid [16S]), two partial mitochondrial genes (cytochrome b [cyt-b] and NADH-dehydrogenase subunit 2 [ND2]) and one partial nuclear gene (oocyte maturation factor [c-mos]). The specific primer pairs used can be found in Table 1 . Each amplification was conducted with a PCR mixture to the total volume of 25 µl containing 12.5 μl TopTaq Mastermix (Ampliqon; containing 2× master mix, 1.5 mM MgCl2, 0.4 mM dNTPs, and Ampliqon Taq DNA polymerase), 2 µl forward primer (10 µM), 2 µl reverse primer (10 µM), 6.5 µl de-nucleated water and 2 µl genomic DNA (20–50 ng/µl). The cycling profile for all the genes was as follows: initial denaturing step at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 50–60 °C for 45 s, and 72 °C for 45 s, with a final extension at 72 °C for 8 min. The prepared PCR products were purified and sequenced at Macrogen Corp. (Amsterdam, Netherlands) with the forward primers only.
10
Touchdown PCR for Microsatellite Amplification
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The touchdown PCR was performed with Taq DNA polymerase (Ampliqon A/S, Stenhuggervej 22, Odense M, Denmark) as follows; denaturing at 95 °C for 1 min, followed by 10 cycles of denaturing at 95 °C for 30 sec, primer annealing at 65 °C for 30 sec (-2 °C in 2 cycles), and extension at 72 °C for 30 sec, and then, 35 cycles of denaturing at 95 °C for 30 sec, primer annealing at 55 °C for 30 sec, and extension at 72 °C for 30 sec, and final extension at 72 °C for 1 min. The amplified samples were electrophoresed in 12% polyacrylamide gels in TBE solution (89 mM Tris, 89 mM boric acid and 2 mM EDTA), and then, stained with ethidium bromide. The stained samples were visualized and photographed under ultraviolet lamp. The results of PCR amplification of representative microsatellite markers were shown in Fig. 3.
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