All chemicals were of analytical grade and were purchased from Sigma-Aldrich (USA), Qualigens (India), Merck (India), Himedia (India) and SRL (India). LB media was purchased from Himedia (India). Ni+2-NTA resin, Protein G beads and Taq polymerase were purchased from Merck-Millipore (USA). cDNA synthesis kit was purchased from Thermo-Fisher (USA). Biorex-70 resin was purchased from Bio-Rad (USA). Cell culture chemicals were purchased from Sigma-Aldrich (USA) while cell culture plasticware was from Corning (USA). SYBR Green mix was purchased from Kapa Biosystems (USA).
Taq polymerase
Manufactured by Merck Group
Sourced in United States, Germany, India, United Kingdom, Macao
Taq polymerase is a thermostable DNA polymerase enzyme derived from the thermophilic bacterium Thermus aquaticus. It is commonly used in the polymerase chain reaction (PCR) technique for the amplification of DNA fragments.
Automatically generated - may contain errors
Lab products found in correlation
Aligner v9, by CodonCode (9 mentions)
Dntps, by Thermo Fisher Scientific (6 mentions)
Dntps, by Merck Group (4 mentions)
Pcr buffer, by Merck Group (4 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Premix d, by Illumina (3 mentions)
Mgcl2, by Merck Group (3 mentions)
Dntp mix, by Merck Group (2 mentions)
Cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Exosap it express, by Thermo Fisher Scientific (2 mentions)
Simpliamp thermocycler, by Thermo Fisher Scientific (2 mentions)
Rotor gene 6000, by Qiagen (2 mentions)
Mastercycler gradient, by Eppendorf (2 mentions)
Quickextract dna extraction solution, by Illumina (2 mentions)
Geltrex coated, by Thermo Fisher Scientific (2 mentions)
Herculase fusion polymerase, by Agilent Technologies (2 mentions)
Novex 4 20 tbe gels, by Thermo Fisher Scientific (2 mentions)
Puromycin, by Merck Group (2 mentions)
Custom pcr master mix, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
79 protocols using taq polymerase
1
Protein Purification and Characterization
2
Molecular Cloning Reagents Protocol
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Taq polymerase, dNTP mix, ultrapure water, loading buffer, and Tris-EDTA buffer were purchased from Merck (Darmstadt, Germany); rDNA and LSU primers were purchased from Macrogen Korea; and protease inhibitor cocktail IV, agarose, chloroform, isoamyl alcohol, protease inhibitor cocktail, acrylamide, and methanol were purchased from Sigma Chemical Co., Ltd. (St. Louis, MO, USA).
3
Pathogenic Vibrio Species Detection
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PCR was performed with purified genomic DNA as template from all the isolated enteropathogenic Vibrios for the detection of the species specific toxin genes associated with pathogenicity of the particular Vibrio species. The reaction mixtures consisted of 10X PCR reaction buffer, 2.5mM DNTP mixture, 1.5 mmol l-1 MgCl2, 100 nmol l-1of primer, 1 U Taq polymerase (Genie, Merck) and 2 μl of template DNA. Nucleotide sequence of the primers and the conditions of the PCR are presented in Table 1 .
4
Dermatophyte Genomic DNA Extraction and Multiplex PCR
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The genomic DNA of the dermatophytes was extracted using the Qiagen DNeasy Plant Tissue Kit (QIAGEN, Germany). Following quality confirmation by Nanodrop (Eppendorf, Germany), specific primers for target genes were designed by the Gene runner software and blasted on the NCBI website to confirm specificity. Multiplex PCR was used to detect isolates harboring Mep1-5, Erg11, 24, 26, ScpA, B, and NPII genes. The reaction mixture of PCR amplification was adjusted to 50 μl, which included 50 ng of genomic DNA solution, 10× PCR buffer, 0.6 U of Taq polymerase (Merck, Germany), 0.1 mM of dNTPs, and 0.5 mM of the primer (Table 1 ). The temperature steps of the PCR reaction were performed as follows: initial denaturation step at 95°C for 5 min in 35 cycles, including 95°C denaturation for 30 sec, and primer binding at 56°C for 30 sec. The amplification step was performed at 72°C for 1 min; after 35 cycles, the final amplification step was carried out at 72°C for 10 min. PCR products were electrophoresed on 1.5% agarose gel in the presence of positive and negative controls, stained with erythrogel, and photographed by a gel documentation device.
5
CYP3A5*3/*3 ABCB1 Methylation Analysis
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Genomic DNA (500 ng) extracted from the 23 CYP3A5*3/*3 genotype donor livers was transformed by sodium bisulfite using the EZ DNA Methylation Kit (Zymo Research, Orange County, CA, USA) and further purified using the Wizard DNA Clean‐up System (Promega, Madison, WI, USA). The sequences of the primers used for the ABCB1 DNA methylation analysis are shown in Table 1 . Each polymerase chain reaction (PCR) consisted of 100 ng of DNA converted by sodium bisulfite, 100 pM deoxyriboside triphosphate, 10 pM positive/reverse primers and 1 unit of Taq polymerase (Merck KGaA, Darmstadt, Germany), which resulted in a final volume of 25 μL. After initial denaturation at 95°C for 5 minutes, amplification was performed for 40 cycles at 95°C for 30 seconds, 60°C for 30 seconds and 72°C for 30 seconds, followed by a final elongation step at 72°C for 5 minutes. PCR products were analysed by nondenatured 6% polyacrylamide gel electrophoresis, following staining with ethidium bromide.
In addition, genomic DNA was extracted from cells treated with the methylation inhibitor, 5‐Aza‐2‐DC and changes in ABCB1 DNA methylation were also evaluated by pyrosequencing.
In addition, genomic DNA was extracted from cells treated with the methylation inhibitor, 5‐Aza‐2‐DC and changes in ABCB1 DNA methylation were also evaluated by pyrosequencing.
6
Genotyping of Interleukin 18 Polymorphism
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Genomic DNA was isolated from fresh blood by salting out procedure [10 (link)] stored at -20°C. Interleukin 18-137G/C (rs187238) gene polymorphism was carried out by Allele specific step down PCR method. Briefly, two complementary reactions were established for each allele along with the common reverse primer (5’-AGGAGGGCAAAATGCACTGG-3’ ) and two allele specific primers (5’-CCCCAACTTTTACGGAAGAAAAC -3’ and 5’-CCCCAACTTTTACGG AAGAAAAG -3’ ) respectively and control forward primer (5’-CCAATAGGACTGATTATT CCGCA-3’ ) (internal positive amplification control).
Each amplification reaction contained 5 μl of 10X buffer containing 15 mM MgCl2, 0.5 μl of 10 pm of each primer, 0.25 μl of 10mM dNTPs (Merck), 0.2 U of Taq polymerase (Merck) and 2 μl of DNA.
Reactions were carried out in a Bio-Rad Thermo Cycler (Bio-Rad Laboratories, Hercules, CA, USA). Initial denaturation at 94°C for 2 minutes, denaturation at 94°C for 20 seconds, annealing at 64.5°C for 40 seconds and extension at 72°C for 70 seconds as the first step followed by 25 cycles of denaturation at 94°C for 20 seconds, annealing at 61°C for 40 seconds and extension at 72°C for 40 seconds. The final extension was performed at 72°C for 5 minutes.
All PCR products were visualized under UV light on ethidium bromide-stained 2% agarose gels. An amplification product of 261 bp was detected for G & C and 446 bp for control forward primer.
Each amplification reaction contained 5 μl of 10X buffer containing 15 mM MgCl2, 0.5 μl of 10 pm of each primer, 0.25 μl of 10mM dNTPs (Merck), 0.2 U of Taq polymerase (Merck) and 2 μl of DNA.
Reactions were carried out in a Bio-Rad Thermo Cycler (Bio-Rad Laboratories, Hercules, CA, USA). Initial denaturation at 94°C for 2 minutes, denaturation at 94°C for 20 seconds, annealing at 64.5°C for 40 seconds and extension at 72°C for 70 seconds as the first step followed by 25 cycles of denaturation at 94°C for 20 seconds, annealing at 61°C for 40 seconds and extension at 72°C for 40 seconds. The final extension was performed at 72°C for 5 minutes.
All PCR products were visualized under UV light on ethidium bromide-stained 2% agarose gels. An amplification product of 261 bp was detected for G & C and 446 bp for control forward primer.
7
Genotyping of Arg399Gln Polymorphism
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Genomic DNA was isolated by phenol–chloroform method [31 ] from uncoagulated blood samples collected in EDTA–coated tubes. Genotyping (of Arg399Gln) was carried out by PCR amplification, followed by RFLP using MspI (NEB, England). The amplification reaction was performed in Applied Biosystems 9902 Thermal Cycler with a reaction volume of 15 μl containing 0.5 μl 100 pmol primer (IDT, USA, Fwd: 5’-GTTGTGCTTTCTCTGTGTCCA-3’ ; Rev: 5’-TCCTCCAGCCTTTTCTGATA-3’ ), 0.5μl 10mM dNTP mix (Merck), 0.5 μl 10x Taq buffer A (Merck), 0.5 μl Taq Polymerase (Merck) and 1μl template DNA (50 ng). The amplification protocol followed was: initial denaturation at 94°C (8 min), followed by 35 cycles of denaturation at 94°C (30 sec), annealing at 61°C (30 sec), initial extension at 72°C (30 sec), and final extension at 72°C (10 min). The amplified products were visualised in 1.5% agarose gel (Fig 1 ). The restriction digestion (MspI) was performed as per the manufacturer’s protocol, and the digested amplicon products (627 bp for Gln399Gln, 383 + 244 bp for Arg399Arg) were resolved on 2.8% agarose gel (Fig 2 ). The genotypic analysis performed with the Restriction Fragment Length Polymorphism (RFLP) method was followed by sequencing and proved to be consistent with that of the bands observed; the sequence information was deposited in NCBI GenBank, and the Accession Numbers were obtained.
8
Targeted Sequencing of IDH Mutations
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Genomic DNA was isolated from peripheral blood or bone marrow samples using standard phenol-chloroform method. Exon 4 of IDH1 and IDH2 were amplified by polymerase chain reaction using the primer pair; IDH1F (5'-AGCTCTATATGCCATCAC TGC-3'), IDH1R (5'-AACATGCAAAATCACATTATTGCC-3'), and IDH2F (5'-AATTTT AGGACCCCCGTCTG-3'), and IDH2R (5'-CTGCAGAGACAAGAGGATGG-3') (Chotirat et al., 2012) . The PCR performed in a 25 µl reaction containing 50 ng of genomic DNA, 1.5 mM MgCl 2 , 0.2 mM dNTPs, 12.5 pmol of each oligonucleotide primers, and 0.5 units of Taq polymerase (Merck, Germany). The PCR condition consisted of an initial denaturation step at 98˚C for 30 seconds, followed by 35 cycles at 98˚C for 10 Sec, 62˚C for 30 Sec, 72˚C for 30 Sec and a final step at 72˚C for 5 min for IDH1 and initial denaturation step at 98˚C for 30 seconds, followed by 35 cycles at 98˚C for 10 Sec, 61˚C for 30 Sec, 72˚C for 30 Sec and a final step at 72˚C for 5 min for IDH2. Then the PCR products were analyzed for single-strand mobility patterns and the samples showing altered mobility were sequenced in both sense and antisense directions to confirming the mutations. The sequences were compared to the wild type IDH1 and IDH2 cDNA (GenBank Accession number, NM_005896.2 and NM_002168.2, respectively).
9
Screening for NPM1 Mutations in Cancer
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For the screening of the NPM1 mutation, we amplified genomic DNA corresponding to exon 12 of NPM1 by PCR using the primers NPM1F (5'-CTAGAGTTAACTCTCTGGTGG-3') and NPM1R (5'-CCTGGACAACATTTATCAAAC-3') as previously reported (Ahmad et al., 2009) . The Briefly PCR reaction was performed in a 25 µl reaction containing 50 ng of genomic DNA, 1.5 mM MgCl 2 , 0.2 mM dNTPs, 10 pmol of each oligonucleotide primers, and 0.5 units of Taq polymerase (Merck, Germany). The PCR condition consisted of an initial denaturation step at 95˚C for 5 min followed by 35 cycles at 94˚C for 30s, 57˚C for 1 min, 72˚C for 1 min and a final step at 72˚C for 10 min. Then the PCR products were analyzed for single-strand mobility patterns and the samples showing altered mobility were sequenced for confirming the mutations. The sequences were compared to the wild type NPM1 cDNA (GenBank Accession number, NM_002520).
10
PCR Amplification of IL17A Gene
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The DNA fragment of interest from the IL17A gene was amplified by PCR using specific primers: Forward primer: 5′-TCT CCA TCT CCA TCA CCT TTG-3′ and reverse primer: 5′-GTC CAA ATC AGC AAG AGC ATC-3′ under the following specific PCR conditions: initial denaturation (94 °C/5 min), denaturation (94 °C/59 s), annealing (57 °C/59 s), extension (72 °C/59 s), and final extension (72 °C/10 min). The PCR reaction mixture (25 μL) consisted of buffer with MgCl2 (concentration/volume; 10X/2.7 μL); MgCl2 (20 μM/0.2 μL, added additionally); dNTP mixture (10 μM/2 μL; 0.5 each); gene-specific primers, specifically forward (10 μM/0.7 μL) and reverse (10 μM/0.7 μL); Taq polymerase (2 Units/0.4 μL); nuclease-free water (15.3 μL); and DNA template (50 ng/μL/2 μL). The genes were amplified on Thermocycler (Applied Biosystems, Life Technologies, Model #: 9902, Made in Singapore). The PCR amplicon of the IL17A gene was run on a 2% agarose gel and subsequently visualized in the gel-documentation system. The buffer, MgCl2, dNTPs, and Taq polymerase were procured from Sigma-Aldrich, St. Louis, MO, USA, however, primers were purchased from Merck, Germany.
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