The expression levels of RPL13A (a reference gene), VEE-hOct4, VEE-hKlf4, VEE-hSox2, VEE-hGlis1, Rex1, Oct4, and Nanog mRNA in ciPSCs were analyzed by RT-PCR. All the cDNA samples of ciPSC-like colonies were stored at −80 °C until PCR analysis. Total RNA was extracted from putative ciPSCs using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). Complementary DNA (cDNA) was synthesized using Moloney murine leukemia virus (MMLV) reverse transcriptase (Invitrogen) and random primers (Invitrogen). All the procedures were performed in accordance with the manufacturer’s instructions. RT-PCR analysis was conducted using cDNA from putative ciPSCs. cDNA was amplified in a 20 μL PCR mixture consisting of 10 pmol of forward and reverse primers, 2 units of Taq polymerase, 2 µL of 10x PCR buffer, 5 pmol of dNTP mixtures (all from iNtRON Biotechnology, SungNam, Korea), and template DNA. The oligonucleotide primer sequences are presented in Table S1 . PCR amplification was performed for 30 cycles of denaturation at 95 °C for 30 s, annealing at 57 °C for 30 s, and extension at 72 °C for 90 s. The reaction products were analyzed on a 1.25% agarose gel pre-stained with RedSafe™ Nucleic Acid Staining Solution (iNtRON Biotechnology).
Taq polymerase
Manufactured by iNtRON Biotechnology
Taq polymerase is a thermostable DNA polymerase enzyme derived from the bacterium Thermus aquaticus. It is a commonly used enzyme in polymerase chain reaction (PCR) techniques for the amplification of DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Random primer, by Thermo Fisher Scientific (1 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Redsafe nucleic acid staining solution, by iNtRON Biotechnology (1 mentions)
Tri reagent, by Molecular Research Center (1 mentions)
Topscript rt drymix, by Enzynomics (1 mentions)
Gel doc 2000, by Bio-Rad (1 mentions)
Reverse transcription system, by Promega (1 mentions)
Dntps, by iNtRON Biotechnology (1 mentions)
Geneamp 9700, by Thermo Fisher Scientific (1 mentions)
Gel documentation system, by Bio-Rad (1 mentions)
Abi prism 7000 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Lightcycler 3, by Roche (1 mentions)
Taqman reverse transcription reagent, by Thermo Fisher Scientific (1 mentions)
7 protocols using taq polymerase
1
Characterization of Induced Pluripotent Stem Cells
2
Gene Expression Analysis of Swiprosin-1 and -2
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Total RNA was isolated with TRI reagent (Molecular Research Center, Cincinnati, OH) and reverse-transcribed using TOPscript RT DryMIX (Enzynomics, Daejeon, Korea). The resulting cDNA was subjected to PCR using Taq polymerase (iNtRON BioTechnology, Seongnam, Korea) with the following primers: 5′-cggcagggatggcttcat-3′ (sense) and 5′-ttggcacccttaacgccc-3′ (antisense) for Swiprosin-1, 5′-tcttcaatccctacaccg-3′ (sense) and 5′-tggaaaatgagcaggaac-3′ (antisense) for Swiprosin-2, and 5′-tcaccatcttccaggagcga-3′ (sense) and 5′-cacaatgccgaagtggtcgt-3′ (antisense) for glyceraldehyde-3-phosphate dehydrogenase (Gapdh).
3
Quantifying IFNAR2 and BAX mRNA in 5-FU-treated Ishikawa cells
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To analyze the mRNA levels of IFNAR2 and BAX in the Ishikawa cells following 5-FU treatment, RT-PCR was performed. The reactions contained cDNA template, Taq polymerase (iNtRON Biotechnology), dNTP, 10X PCR buffer (iNtRON Biotechnology), and reverse and forward primers (Table II). PCR amplification was performed for 30 cycles of denaturation at 95˚C for 30 sec, annealing at 58˚C for 30 sec, and extension at 72˚C for 30 sec. The PCR products were loaded onto a 1.5% agarose gel prestained with ethidium bromide (EtBr; Sigma-Aldrich). The bands were analyzed with Gel Doc 2000 software (Bio-Rad Laboratories Inc., Hercules, CA, USA). GAPDH was used as an endogenous control for normalization. results of the animal experiments followed by Tukey's multiple comparison test. Data were expressed as the mean ± standard error of the mean (SEm) and P-values <0.05 were considered statistically significant.
4
RNA Extraction and Gene Expression Analysis
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Total RNA from cell lines was extracted with TRIzol reagent (Invitrogen). Reverse transcription was performed using the reverse transcription system (Promega, USA). PCR primers for amplification were as follows: Snail, forward 5′-GAGGACAGTG GGAAAGGCTC-3′, reverse 5′-TGGCTTCGGATGTGCATCTT-3′; Slug, forward 5′-GAACTCACACGGAGAAG-3′, reverse 5′-ACACAGCAGCCAGATTCCTC-3′; vimentin, forward 5′-AATG GCTCGTCACCTTCGTGAAT-3′, reverse 5′-CAGATTAGTTTC CCTCAGGTTCAG-3′; E-cadherin, forward 5′-GGAAGTCAGTT CAGACTCCAGCC-3′, reverse 5′-AGGCCTTTTGACTGTAAT CACACC-3′; GAPDH, forward 5′-GAGAAGGCTGGGGCTCAT TT-3′, reverse 5′-AGTGATGGCATGGACTGTGG-3′. PCR was performed using Taq polymerase (iNtRON Biotechnology Inc., Korea). PCR was initiated by incubating the samples at 95°C for 5 min, followed by 35 cycles of 40 s denaturation at 95°C, 40 s annealing at 55.4°C (for Snail), 57.5°C (for Slug and GAPDH), 58°C (for vimentin), 58.7°C (for E-cadherin) and 5 min elongation at 72°C. Samples were analyzed by electrophoresis on 1.2% agarose gels containing 0.002% nucleic acid staining solution (RedSafe™; Biotechnology Inc., Korea).
5
PCR Profiling of Pseudomonas aeruginosa Virulence
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The PCR was carried out by using primers targeting (oprI, toxA and lasB) virulence genes [10] ., the PCR assay included 25 μl final reaction mixture which consisted 5 μl of (taq polymerase, reaction buffer., MgCl and DNTPs) (Intron biotechnology, Korea) and 2 μl of each 10 P mol forward and reverse primers specific for these virulence genes, 5 μl from DNA template and 13 μl from (ddH 2 O) to complete the volume to 25 μl final reaction mixture .The reaction tubes were cycled in thermal cycler machine (Techne, England). The PCR program was performed with an initial denaturation for 5 min at 94°C, then 35 cycles of denaturation for 1min at 94°C, annealing for 1min at 58°C and 60°C for lasB gene, extension for 1 min at 72°C and final extension for 5 min at 72°C. The amplified products were resolved in %2 agarose gel electrophoresis stained with ethidium bromide and visualized under UV light [10] . Primers used for amplification of virulence genes of P. aeruginosa isolates as in Table-1.
6
Rapid Amplification of Polymorphic DNA
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Random primers used for RAPD in this study are shown in Table 3 and synthesized by Bionix (Korea). The reaction was performed as described by Kern et al. [27 (link)] with some modifications. The reaction mixture (a final volume of 20 μl) contained 1× buffer, 2.5 mM MgCl2, 200 μM deoxynucleoside triphosphates, 2 μM primer, 1.25 U Taq polymerase (Intronbiotechnology, Korea), and 1 μl of genomic DNA prepared as described above. The amplification was performed in a GeneAmp 9700 thermocycler (Applied Biosystems, FUSA) with the following thermal cycling conditions: pre-denaturation at 94°C for 2 min, 40 cycles of 94°C for 15 sec, 35°C for 30 sec, and 72°C for 2 min, followed by 10 min at 72°C. After the reaction, 10 μl of each PCR product was analyzed on a 1%agarose gel. These gels were visualized with a gel documentation system (Bio-Rad, Italy).
7
Quantitative RT-PCR for Gene Expression
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qRT-PCR assays were conducted as previously reported 14 (link),15 (link). The total RNA was isolated from cells using RNA-stat reagent (Iso-Tex Diagnostics, Friendswood, TX, USA), according to the manufacturer’s instructions, before the extracted RNA was reverse-transcribed using Taqman Reverse Transcription Reagents (Applied Biosystems). The cDNA was synthesized using human-specific primers and Taq polymerase (iNtRON Biotechnology, Sungnam, Korea). The DNA levels were quantitatively assessed using an ABI PRISM 7000 Sequence Detection System (Applied Biosystems). qRT-PCR cycling conditions were 10 min. at 95°C, followed by 40 cycles of 15 sec. at 95°C and 1 min. at 60°C. After normalization to GAPDH, the relative expression levels of the target gene in the experimental samples were determined using the formula Rel Exp = 2−ΔCT (fold difference), where ΔCt = (Ct of target gene) − (Ct of control gene, GAPDH). The number of PCR cycles was measured using Lightcycler 3.5 software (Roche Molecular Biochemicals, Indianapolis, IN, USA).
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