Taq dna polymerase

Manufactured by Sinaclon

Taq DNA polymerase is an enzyme derived from the thermophilic bacterium Thermus aquaticus. It is a DNA-dependent DNA polymerase, responsible for the synthesis of DNA strands complementary to a template DNA sequence.

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Lab products found in correlation

Sybr safe dna gel stain, by Thermo Fisher Scientific (1 mentions) Pcr buffer, by Sinaclon (1 mentions) Agarose gel, by Sinaclon (1 mentions) Veriti thermal cycler, by Thermo Fisher Scientific (1 mentions) Gel doc xr system, by Bio-Rad (1 mentions) Qiaamp dneasy mini kit, by Qiagen (1 mentions) Pucm t vector, by Bio Basic (1 mentions) Dntp mix, by Sinaclon (1 mentions) Mboii restriction enzyme, by Thermo Fisher Scientific (1 mentions) Psti restriction enzyme, by Thermo Fisher Scientific (1 mentions) 100 bp dna ladder, by Thermo Fisher Scientific (1 mentions)

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Taq polymerase

13 protocols using taq dna polymerase

Detecting Virulence Genes in Pseudomonas aeruginosa

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Some strains of Pseudomonas aeruginosa were selected for detection oprL, toxA and phzM. DNA was extracted by boiling method as described by Reischl et al. (2002) . The three sets of primers (Table 1) were used for detection of virulence genes. Each PCR reaction was done in a total volume of 20µl as follows: 2 µl of template DNA, 0.6 µl MgCl2, 0.4 µl of each primer, 0.2 µl dNTP, 2µl of 10 x PCR buffer, 0.5 µl of Taq DNA polymerase (5U/µl) (SinaClon, Tehran, Iran) and 13.9 µl of Milli-Q water. The PCR condition was an initial denaturation at 95ºc for 5 minutes, then processed into 35 cycles each one was denaturation at 95ºc for 30 second, annealing at 55 ºc for 35 seconds and extension at 72 ºc for 30 second. A PCR reaction without any DNA was utilized as a negative control, while a reference strain of Pseudomonas aeruginosa gladly given by the Animal Health Institute in Giza, Egypt, was utilized as positive control.
Table 1. Primers used for the amplification of different virulence genes among Pseudomonas aeruginosa isolates.

Abd-El-Maogoud H.A., Edris A.B.M., Mahmoud A.H., & Maky M.A. (2021). Occurrence and characterization of Pseudomonas species isolated from Fish Marketed in Sohag Governorate, Egypt. SVU- International Journal of Veterinary Sciences, 4(2), 76-84.

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Multiplex PCR for Salmonella spp. Detection

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All samples were subjected to m‐PCR analysis to identify Salmonella spp., S. enteritidis and S. typhimurium. The sequences of the primer sets are shown in Table 1. The m‐PCR was performed with the addition of 2 μL of DNA extracted to the PCR mix containing 1× PCR buffer (Sinaclon), 0.4 mM of the dNTP mix (Bio Basic), 0.5 μM of each primer, 2.0 mM MgCl₂, 12.25 μL of nuclease‐free water, and 1.25 U of Taq DNA polymerase (Sinaclon) to a final reaction of 25 μL. The thermo‐profile was 94°C/2 min for initial denaturation, followed by 35 cycles of 94°C/15 s, 58°C/30 s and 72°C/1 min, and a final extension at 72°C/5 min. Finally, 10‐μL aliquots of PCR products were electrophoresed on a 1% agarose gel and stained with SYBR Safe DNA Gel‐Stain (Thermo Fisher Scientific). S. typhimurium ATCC 14028, S. enteritidis ATCC 13076, Salmonella heidelberg ATCC 8326, Salmonella typhi ATCC 27870, and Escherichia coli ATCC 25922 were used as quality control strains.

Yousefi Amin A., Oshaghi M., Habibi S., Bashashati M., Fallah Mehrabadi M.H, & Safavieh S.S. (2024). Prevalence and antimicrobial susceptibility of Salmonella enteritidis and Salmonella typhimurium isolated from hen eggs and quail eggs in Karaj, Iran. Veterinary Medicine and Science, 10(3), e1475.

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LEPR Gene Polymorphisms Analysis via PCR-RFLP

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Analysis of LEPR gene polymorphisms (R223Q and P1019P) were done using
polymerase chain reaction (PCR) amplification, followed by restriction fragment length
polymorphism (RFLP) techniques. PCR assay was performed in a total volume of 25 µl. It was
including 2.5 μl of 10X PCR buffer, 20 pmol of each primer (Table 1), 1.5 mM
MgCl₂ , 0.2 mM dNTPs, 1 Unit Taq DNA polymerase (SinaClonBioScience Co.,
Iran) and 100 ng isolated DNA as PCR template. The defined condition for thermal cycler
included an initial denaturation at 95°C for 2 minutes, 30 cycles of denaturation at 95°C
for 30 seconds, annealing at 60°C (LEPR R223Q)/53°C
(LEPRP1019P) for 30 seconds, extension at 72°C for 40 seconds, and then
a final extension at 72°C for 5 minutes. After amplification, about 10 µl PCR products
were subjected to overnight digestion with 1-2 units of restriction enzymes. Products of
the digestion were visualized by 2% agarose gel stained with GelRED under ultraviolet
light. In the case of R223Q genotyping, BsaWI (Thermo Fisher, USA) at 60°C produced a 279
bp band (AA genotype), 279 bp, 193 bp, and 86 bp bands (AG genotype), or193 bp and 86 bp
bands (GG genotype, Fig.1A). For P1019P genotyping, NcoI (Thermo Fisher, USA) at 37°C
produced a 253 bp band (CC genotype), 253 bp and 223 bp bands (CT genotype) or 223 bp band
(TT genotype, Fig.1B).

Naseri R., Barzingarosi E., Sohrabi M., Alimoradi Y., Cheraghian Fard M, & Jalili C. (2021). The Effect of Leptin Receptor Gene Polymorphisms (R223Q and P1019P) in Susceptibility to Polycystic Ovarian Syndrome in Kurdish Women. International Journal of Fertility & Sterility, 15(2), 123-127.

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COI Gene Amplification for Barcoding

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For PCR amplification of the COI, the 658bp amplicon, the forward and the reverse primers LEP-F (5′-ATT CAA CCA ATC ATA AAG ATA TNG G- 3′) and LEP-R (5′-TAW ACT TCW GGR TGTCCR AAR AAT CA-3′) were used (32 (link)). The PCR was conducted in 25µl total volume, each containing 2.5µl 10x PCR buffer, 2μL 50mM MgCl2, 0.5µl dNTPs, 3µl DNA template, 10 pico-mole forward and reverse primers (0.5µl for each), 0.5µl Taq DNA polymerase (Sinaclon, Iran) and 15.5µl ddH₂O. The PCR cycling conditions set in the program were as follows: initial denaturation at 94 °C for 5min followed by 35 cycles of 94 °C for 30sec (denaturation), 42 °C for 30sec (annealing), 72 °C for 45sec (extension) and a final extension step of 72 °C for 2min. PCR products were analyzed by electrophoresis in 1.5% agarose gel to confirm that the samples contained a single band. Then, stained with safe DNA stain gel and visualized with UV-Transilluminator (BTS-20M, Japan). Finally, Purified PCR products were sent to SinaClon Company (Tehran, Iran) for sequencing.

Samiei A., Tavassoli M, & Mardani K. (2020). The Phylogenetic Analysis of Cimex hemipterus (Hemiptera: Cimicidae) Isolated from Different Regions of Iran Using Cytochrome Oxidase Subunit I Gene. Journal of Arthropod-Borne Diseases, 14(3), 239-249.

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Transgenic Plant DNA Verification

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DNA was extracted from the mature leaves of the seedlings’ tissue and Polymerase Chain Reaction (PCR) was carried out with the above gene specific primers with Taq DNA polymerase (SinaClon BioScience Co., Tehran, Iran) to verify transgenic lines. The following amplification program was used in the PCR amplification: 94 °C for 5 min, followed by 35 cycles of 94 °C for 1 min, 60 °C for 1 min, 72 °C for 1 min and final extension for 10 min at 72 °C.

Sabet M.S., Zamani K., Lohrasebi T., Malboobi M.A, & Valizadeh M. (2018). Functional Assessment of an Overexpressed Arabidopsis Purple Acid Phosphatase Gene (AtPAP26) in Tobacco Plants. Iranian Journal of Biotechnology, 16(1), e2024.

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PCR Amplification of E. coli 23S rRNA

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Amplifications were performed in a gradient thermocycler (Applied Biosystems™ Veriti™ Thermal Cycler, Thermo Fisher Scientific, Waltham, MA, USA) using the primers Eco 2083 F (5′-GCTTGACACTGAACATTGAG-3′) and Eco 2745 R (5′-GCACTTATCTCTTCCGCATT-3′), specific for E. coli and targeted to the 23S rRNA gene [27 ]. The PCR reaction was carried out in a final volume of 25 μL, containing 4–5 μL of template DNA, 2.5 μL of 10× PCR buffer, 0.75 μL of 50 mM MgCl2, 0.5 μL of 10 mM deoxyribonucleoside triphosphates (dNTP), 0.25 μL of 5 U/μL of Taq DNA polymerase (Sinaclon, Tehran, Iran), and 10 pmol of each primer. The PCR amplification was performed in 35 cycles using the following conditions: initial denaturation at 94 °C for 5 min; denaturation at 94 °C for 1 min, annealing at 57 °C for 1 min, extension at 72 °C for 2 min; and final extension at 72 °C for 7 min. E. coli strain ATCC 25922 and distilled water were used as positive and negative standard controls, respectively. PCR products were analyzed by electrophoresis on 2% agarose gel (Sinaclon, Tehran, Iran) containing ethidium bromide (0.5 μg/mL) under ultraviolet (UV) light. PCR gels were digitally captured by GEL DOC XR System (Bio-Rad, Hercules, CA, USA).

Pajohi Alamoti M., Bazargani-Gilani B., Mahmoudi R., Reale A., Pakbin B., Di Renzo T, & Kaboudari A. (2022). Essential Oils from Indigenous Iranian Plants: A Natural Weapon vs. Multidrug-Resistant Escherichia coli. Microorganisms, 10(1), 109.

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Plant DNA Extraction and PCR Amplification

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The genetic material was extracted using the QIAampDNeasy Mini Kit (QIAGEN, Germany) that works based on silica gel membrane technology which allowed an efficient recovery ofcomplete DNA from plant tissues. DNA was extracted from each of the samples between 3-5 times.
All PCR amplification runs were performed using an ABI Simpliamp System (Life Technologies, USA). Each amplification reaction contained 1X reaction buffer, 0.1-0.4 μM of each primer (Table S2); 1 U Taq DNA Polymerase (Sinaclon, Iran), 1.5-3 mM MgCl2, 0.20-0.25mM each dATP, dCTP, dGTP, and dTTP (Sinaclon, Iran). Either 2 or 5μ LDNA was added to 15 or 18 μl prepared Master mixes. All PCR reactions were performed based on Tables S3 andS4.

Sepahian N., Noourmohammadi Z., Sheidai M., & Zamanizadeh H.R. (2021). Authentication, genetic fingerprinting and assessing relatedness of rice (Oryza Sativa) genotypes by SSR molecular markers. Caryologia, 74(1), 13-22.

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Screening for Carbapenemase Genes in Bacteria

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Bacterial DNA was released from whole microorganisms by boiling. Bacteria were harvested from 1 ml of an overnight broth culture, suspended in 200 μl of sterile water, and incubated at 100°C for 10 min. Following centrifugation of the lysate, a 150 μl sample of the supernatant was stored at −20°C as a template DNA stock.[11 (link)] The primers used in this study are shown in Table 1.[8 (link)] Each reaction mixture (15 μl) contained 2 μl of DNA, 0.5 mM of each primer, 1 U of Taq DNA polymerase (Sinaclon), the four deoxynucleoside triphosphates (each at 200 μM) and 1.5 mM MgCl₂. PCR was performed under predenaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 45 s, at each specific annealing temperature [Table 1] for 30 s, and 72°C for 45 s ending with a final extension step at 72°C for 5 min. The PCR product was run and visualized on 1.5% agarose gels then were stained with ethidium bromide. PCR screening was done for the carbapenemase-encoding genes including blaOXA-23-like and blaOXA-24-like. PCR analysis was performed using the primers described previously.[12 (link)]

Mohajeri P., Sharbati S., Farahani A, & Rezaei Z. (2016). Evaluate the frequency distribution of nonadhesive virulence factors in carbapenemase-producing Acinetobacter baumannii isolated from clinical samples in Kermanshah. Journal of Natural Science, Biology, and Medicine, 7(1), 58-61.

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Identification of Lactobacillus Strains from Newborns

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Lb. plantarum 34-5 and Lb. fermentum 89-1 were isolated from the faeces of healthy newborns. Identification of these two strains was performed with 16S rDNA gene sequencing. In brief, genomic DNA was extracted according to a previously described method [8] (link). The PCR primer sequences were as follows: forward primer 5′-CTCGTTGCGGGACTTAA-3′, and reverse primer 5′-GCAGCAGTAGGGAATCTTC-3′ (Bioneer, Korea). The reaction mixture consisted of 3 pmol primers, 1.5 mM MgCl₂, 0.2 mM dNTPs, 2 μL of genomic DNA, 5 μL 10× PCR buffer and 1.5 U of Taq DNA polymerase (Sinaclon, Iran) in a final volume of 50 μL. The PCR program started with an initial denaturation at 94°C for 2 minutes, followed by 30 cycles of 94°C for 30 seconds, 53°C for 1 minute and 72°C for 1 minute [12] (link). PCR products were separated by agarose gel electrophoresis (1.5% w/v) and visualized by staining with ethidium bromide. The PCR products of strains were sent to a sequencing company (Bioneer, Korea), and the 16S rDNA sequences were compared to known sequences in GenBank using the Basic Local Alignment Search Tool (BLAST; http://www.ncbi.nlm.nih.gov/blast).

Soltan Dallal M.M., Davoodabadi A., Abdi M., Hajiabdolbaghi M., Sharifi Yazdi M.K., Douraghi M, & Tabatabaei Bafghi S.M. (2016). Inhibitory effect of Lactobacillus plantarum and Lb. fermentum isolated from the faeces of healthy infants against nonfermentative bacteria causing nosocomial infections. New Microbes and New Infections, 15, 9-13.

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Molecular Identification of Probiotic Strains

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Lastly, the results of the conventional methods (above) for identifying probiotic strains were confirmed by a molecular method. The identification of the selected isolates with acid and bile resistance was confirmed by 16S rDNA sequence analysis. Genomic DNA was extracted according to a previously described method [16 (link)]. The PCR primer sequences were as follows: forward primer, 5'-CTCGTTGCGGGACTTAA-3' and reverse primer, 5'-GCAGCAGTAGGGAATCTTC-3' (Bioneer, Korea) [17 (link)]. The reaction mixture consisted of 3 pmol of primers, 1.5 mM MgCl₂, 0.2 mM dNTPs, 2 µl of genomic DNA, 5 µl 10X PCR buffer and 1.5 U of Taq DNA polymerase (Sinaclon, Iran) in a final volume of 50 µl. The PCR program started with an initial denaturation at 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 53°C for 1 min and 72°C for 1 min. PCR products were separated by Agarose gel electrophoresis (1.5% w/v) and visualized by staining with ethidium bromide. The PCR products of strains were sent to a sequencing company (Bioneer, Korea) and the 16S rDNA sequences were compared with known sequences in GeneBank using BLAST (http://www.ncbi.nlm.nih.gov/blast).

Soltan Dallal M.M., Zamaniahari S., Davoodabadi A., Hosseini M, & Rajabi Z. (2017). Identification and characterization of probiotic lactic acid bacteria isolated from traditional persian pickled vegetables. GMS Hygiene and Infection Control, 12, Doc15.

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