Escherichia coli isolate DNA was extracted by the simple boiling method. E. coli isolates were cultured on MacConkey agar (Merck, Germany) and incubated at 37°C. After 24 hours, 1 - 5 colonies were suspended in TE buffer, and suspensions were boiled for 10 minutes (21 (link)). PCR was performed using gene-specific primers to detect two ESBL-encoding gene types (blaSHV and blaTEM) (21 (link)). The following conditions were used: 2 minutes at 95°C, followed by 35 cycles of 1 minute at 91°C, 30 seconds at 52°C, and 45 seconds at 72°C, and 1 minute at 72°C and 5 minutes at 72°C for the final extension (19 ). Each 25 µL of reaction mixture contained 2.5 µL of 10 × PCR Buffer, 1.5 mM MgCl2 (25 mM), 320 µM dNTPs (10 mM), 500 nM of each primer (10 pm/µL), 50 ng template DNA, and 0.5 U/µl Taq DNA polymerase (CinnaGen, Iran). Pure water was added for a final volume of 25 µl. K. pneumoniae ATCC 7881 containing the blaSHV and blaTEM genes (Pasteur Institute of Iran, Tehran) was used as a positive control.
Taq dna polymerase
Manufactured by CinnaGen
Sourced in United States
Taq DNA polymerase is an enzyme derived from the thermophilic bacterium Thermus aquaticus. It is a heat-stable DNA polymerase commonly used in the polymerase chain reaction (PCR) process to amplify DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Mgcl2, by CinnaGen (6 mentions)
Pcr buffer, by CinnaGen (5 mentions)
Dntps, by CinnaGen (5 mentions)
Gel imager, by Thermo Fisher Scientific (3 mentions)
Abi3700xl dna sequencer, by Thermo Fisher Scientific (2 mentions)
Agarose gel, by Merck Group (2 mentions)
Ethidium bromide, by Merck Group (2 mentions)
Macconkey agar, by Merck Group (1 mentions)
10 mm dntp, by Thermo Fisher Scientific (1 mentions)
Dna safe stain, by Thermo Fisher Scientific (1 mentions)
Accuprep gel purification kit, by Bioneer (1 mentions)
Mj mini thermal cycler, by Bio-Rad (1 mentions)
Cinnapure dna kit, by CinnaGen (1 mentions)
Gel doc, by Uvitec (1 mentions)
Ethidium bromide, by CinnaGen (1 mentions)
100 bp dna ladder, by CinnaGen (1 mentions)
C1000 thermal cycler, by Bio-Rad (1 mentions)
Gene atlas thermal cycler, by Astec (1 mentions)
Pcr purification kit, by Bioneer (1 mentions)
Genomic dna purification kit, by Thermo Fisher Scientific (1 mentions)
56 protocols using taq dna polymerase
1
Screening for ESBL-encoding genes in E. coli
2
PCR for Gene Amplification and Analysis
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PCR technique was performed. Primers were developed for each gene using Primer 3. The primers used for DNA amplification, as follows: 5′-AGACATGCCGTGGATACAAA0-3′ for the forward and 5′- AGTGCTACCTGCGGAAACC -3′ for the reverse primers. The final optimized PCR reaction consisted of 0.5 μl MgCl2 (100 mM), 0.5 μl dNTP (10 mM), 0.2 µl (1 unit) Taq DNA polymerase (Cinnagen, Iran), 1 µl of each primer (10 pmol) (Alpha DNA, Canada), 2.5 µl PCR buffer (10 X), and 0.5 μl of DNA template (100 μg/ml) in a total volume of 25 μl with double distilled water. DNA amplification was carried out with a thermocycler (Quanta Biotech, England), PCR amplification was performed as follows: one cycle at 95 °C for 300 s, then 30 cycles at 95 °C for 45 s, 56 °C for 45 s, and 72 °C for 60 s and a final extension at 72 °C for 10 min using an initial denaturation step for 5 min at 94 °C (one cycle), followed by 35 cycles of 1 min at 94 °C, 1 min at 50 °C, and 1 min at 72 °C. The amplified products were analyzed by 1.5% (w/w) agarose gel electrophoresis and were visualized on an ultraviolet illumination after staining with ethidium bromide.
3
Rapid Nitrocellulose-Based Assay Development
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In this experimental study, nitrocellulose (NC) membrane (Hi-flow plus), absorbent pad (cellulose fiber sample pad roll), sample pad (cellulose fiber sample pad roll) and conjugate pad (glass fiber conjugate pad rolls) were purchased from Merck Millipore (Germany). Plastic adhesive rigid back were prepared from a local store. Chemicals were acquired from Scharlau (Spain) or Sigma (USA). Taq DNA polymerase was obtained from CinnaGen Co. (Iran). Terminal deoxynucleotidyl transferase (TdT) kit was prepared from Thermo Scientific (USA). Deoxyadenosine triphosphate (dATP) was from Jena Bioscience (Germany). All solutions were prepared with RNAase free water.
4
Molecular Profiling of Helicobacter pylori Virulence Factors
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DNA was extracted from the biopsy specimens by using Genomic DNA purification kit (DNGTM-Plus, CinnaGen Co., Iran) according to the manufacturer's recommendations, and stored at -20ºC. After DNA extraction, polymerase chain reaction (PCR) was performed in a volume of 30 µL reaction volume containing 50 ng of genomic DNA, 3 µL of 10X PCR buffer (CinnaGen, Iran), 1 mM MgCl2, 200 µM of each dNTP (CinnaGen Co., Iran), 0.5 µM of each specific primer and 2 U of Taq DNA polymerase (CinnaGen Co., Iran). Amplification was also performed under the following conditions: for cagA: 35 cycles of 40 sec at 94°C, 1 min at 50°C (C-terminus), 55°C (middle region), or 56°C (N-terminus) and 1 min at 72°C, and for babA2: 35 cycles of 40 sec at 94°C, 1 min at 55°C and 1 min at 72°C. PCR products were visualized by electrophoresis in 1% agarose gel and examined under UV illumination. Primers used for PCR assays of 16S rDNA, cagA and babA2 genes are also listed in Table 1. As controls, amplified fragments of each gene from seven isolates were purified and sequenced with both forward and reverse primers by using BigDye technology on an ABI3700XL DNA sequencer (Applied Biosystems). The BLAST program (http://www.ncbi.nlm. nih.gov) was used to match the nucleotide sequences with the published sequences in GenBank.
5
VNTR Loci Amplification Protocol
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These seven VNTR loci were selected: ms06, ms07, ms09, ms11, ms21, ms23 and ms32. The primer sets for PCR amplification of these VNTR loci were previously reported (Table 1 ) (19 (link)). PCR was performed in 25 μl volume containing 1X PCR buffer (50mmol/L KCL, 10 mmol/L Tris, pH9), 2.5 mmol/L MgCl2, 0.2 mmol/L of each primer with 0.5 U TaqDNA polymerase (CinnaGen Co., Iran), and 3 μl of DNA extract. Cycling conditions for PCR reactions were 93 °C for 5 min, followed by 34 cycles of 93 °C for 30 sec, 55 °C for 1 min, and 72 °C for 1 minute. The PCR products were run on agarose gels, stained with ethidium bromide, and visualized under UV transillumination by gel doc.
6
Cloning miR-129 and 3' UTR Reporters
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The mir-129 gene was amplified by PCR using Taq DNA polymerase (cinnaGen) and specific primers (Table S1 ). Then, it was cloned into a pCDH-CMV-MCS-EF1-cGFP-T2A-puro vector (System Biosciences, USA). The full-length 3′ UTR of the CDK4 gene (1,060th nt to the end of mRNA NM_000075.4) and partial lengths of CDK6 3′ UTR (6,998–11,580 nt of transcript variant 1 mRNA NM_001259.8) and MDM2 3′ UTR (1,593–6,048 nt of transcript variant 3 mRNA NM_001145337.3) were amplified by PCR on genomic DNA using Takara SpeedSTAR HS DNA polymerase (catalog no. RR070A/B) and specific primers (Table S1 ). PCR fragments were cloned into the psiCHECK-2 Dual-Luciferase reporter plasmid separately (Promega, Madison, WI, USA) downstream of the Renilla luciferase gene. The pLenti-III-eGFP scramble vector was used as a negative control for miRNA.
7
Rotavirus Detection via RT-PCR
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As shown in Table 1 , one oligonucleotide primer pair was used to detect rotavirus. The PCR reaction was performed in a final volume of 25 μL containing 4 μL 10× PCR buffer (CinnaGen, Iran), 0.5 μl each 10 pmol/μl primer, 1 μl 10 mM dNTPs (Fermentas, USA), 0.3 μl 500 U/μl Taq DNA polymerase (CinnaGen), 0.5 μl 50 mM MgCl2 (CinnaGen), 11.2 μL H2O and 7.0 µl cDNA template. PCR cycles were carried out in a thermal cycler (Applied Biosystems GeneAmp® verity thermocycler) as follows: an initial denaturation at 95°C for 5 min, 40 cycles of denaturation at 94°C for 3 min, annealing at 55°C for 55 s, elongation at 72°C for 1 min and a final extension step at 72°C for 7 min. The amplification of 569-bp PCR product was considered as positive.
8
Bacterial DNA Extraction and Amplification
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DNA was extracted using the lysis method and was stored at À20 C. 9 Amplification of bacterial DNA was performed using 25 mL volumes containing 5 mL of the template DNA; 0.2 mmol/L of each deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate, and deoxythymidine triphosphate; 1.5 mmol/L MgCl 2 ; 1Â reaction buffer; 0.5 mmol/L of each primer; and 1.2 U of Taq DNA polymerase (CinnaGen Co, Karaj, Iran). The polymerase chain reaction (PCR) products were electrophoresed via a 1.5% agarose gel and visualized with an ultraviolet (UV) transilluminator after ethidium bromide staining.
9
Multiplex PCR for HPV Genotyping
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A 5-plex PCR was performed for identification of HPV-16, 18, 31, 33 and 45. The Multiplex PCR was run in final reaction volumes of 50 μl containing the following reagents: 100 ng of genomic DNA, 50 mM KCl, 10 mM Tris-HCl (pH 8.5), 6 mM MgCl2, 200 μM each of dATP, dCTP, dGTP and dTTP, 50 pmol of each primer and 5 unit of Taq DNA polymerase (CinnaGen Co, Iran). The PCR assay was performed at 95ºC for 6 minutes (pre denaturation) and then for 40 cycles of 94ºC for one minutes, 60ºC for one minutes, 72ºC for minutes, with final extension at 72ºC for seven minutes in a thermal cycler (Mastercycler gradient, Eppendrof, Germany), Negative controls were included in each run contain HPV 54.
10
Genotyping CYP2B6 Genetic Variants
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CYP2B6∗6 (516G > T), CYP2B6∗4 (785A > G), and CYP2B6∗5 (1459C > T) were genotyped using tetra-primer amplification refractory mutation system polymerase chain reaction (ARMS–PCR). The amplification was conducted using 60 ng of extracted genomic DNA, 0.4 U Taq DNA polymerase (CinnaGen), six pmol of each primer, 10X PCR buffer, 0.5 mM dNTP, and 1.5 mM MgCl2. The mixture was initially denatured at 95°C for 3 minutes, followed by 32 cycles of 95°C for 1 minute, 56°C for 1 min, and 72°C for 2 min for CYP2B6 516G > T; 32 cycles of 95°C for 1 minute, 64°C for 50 seconds, and 72°C for 1 minute for CYP2B6 785A > G; 35 cycles of 95°C for 1 minute, 63°C for 1 min, and 72°C for 1 min for CYP2B6 1459C > T; followed by a final extension at 72°C for 10 min. Afterward, amplified fragments were run on a 1.5% agarose gel electrophoresis for 1 hour at 80 volt, ahead of staining using silver nitrate (CinnaGen) [19 (link), 20 (link)]. Table 1 provides information on the primer sequences [6 (link)]. The DNA bands in the agarose gel were visualized under ultraviolet (UV) rays, and images were captured [19 (link), 20 (link)]. Figure 2 schematically summarizes the genotyping process using Tetra-ARMS-PCR for CYP2B6∗6 (516G > T).
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