PCR with primers for hkg (16S rRNA, dnaK, gltA, and glnII) and sym (nodA, nodC, nodD, nodX and nifH) genes were performed in a 30 µl reaction mixture, containing 10 ng template DNA, 10 pM of each primer, 1× buffer for Taq polymerase (Evrogen, Moscow, Russia), 4.5 nM of each dNTP (Helicon, Moscow, Russia) and one unit of Taq polymerase (Evrogen, Moscow, Russia). PCR reactions were performed on a T-100 thermal cycler (Bio-Rad, Hercules, CA, United States) with an initial denaturation at 95 °C for 3 min, 35 cycles of denaturation (30 s at 94 °C) and annealing (30 s at 50–62 °C), an extension of 1 min at 72 °C, and a final extension at 72 °C for 3 min. Primers and their annealing temperatures are listed in Table 1 . PCR products were cleaned of residual enzyme and primers using a silica binding-based protocol [24 (link)]. All amplicons were directly sequenced on an ABI PRISM 3500xL Genetic Analyzer (Applied Biosystems, Waltham, MA, United States) at the Centre for Collective Use of Scientific Equipment’s “Genomic Technologies, Proteomics and Cell Biology” in ARRIAM.
Taq polymerase
Manufactured by Evrogen
Taq polymerase is a thermostable DNA polymerase enzyme derived from the bacterium Thermus aquaticus. It is a crucial component in the Polymerase Chain Reaction (PCR) process, which is a widely used technique in molecular biology, genetics, and genomics for the amplification of DNA fragments.
Automatically generated - may contain errors
Lab products found in correlation
Abi prism 3500xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
Gotaq buffer, by Promega (1 mentions)
Mytaq polymerase, by Meridian Bioscience (1 mentions)
Mytaq buffer, by Meridian Bioscience (1 mentions)
Gotaq polymerase, by Promega (1 mentions)
Encyclo buffer, by Evrogen (1 mentions)
Bactoagar, by Helicon (1 mentions)
Yeast extract, by Helicon (1 mentions)
Peptone, by Helicon (1 mentions)
Oligonucleotides, by Evrogen (1 mentions)
Dna and protein molecular weight markers, by Bio-Rad (1 mentions)
Tri reagent ls, by Merck Group (1 mentions)
Mmlv rt kit, by Evrogen (1 mentions)
Proflex pcr system, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
100 bp dna ladder, by Evrogen (1 mentions)
Mmlv reverse transcriptase, by Evrogen (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Haucl4, by Merck Group (1 mentions)
6 protocols using taq polymerase
1
Multiplex PCR for Microbial Taxonomy
2
Efficient Long-Range PCR with SD Polymerase
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Example 3
Comparison of SD DNA Polymerase with Taq DNA Polymerase in Long PCR
Example 3 illustrates a high efficiency of SD polymerase in PCR amplification. SD polymerase was compared with Taq polymerases from different suppliers like GoTaq polymerase and GoTaq buffer from Promega; MyTaq polymerase and MyTaq buffer from Bioline; and Taq polymerase and Encyclo buffer from Evrogen
An 8 kb fragment of λ DNA was amplified with 2.5, 5, 10 and 15 units of SD or Taq DNA polymerase. Reaction mixture (50 μl) contained: 5 ng λ DNA as template, 0.25 mM dNTP (each), 10 pmol (0.2 μM) of each primer, 1×PCR buffer, and 3 mM MgCl2.
PCR was carried out for 25 cycles: preheating for 2 min at 92° C.; cycling for 30 sec at 92° C., 30 sec at 60° C. and 2 min 40 sec (20 sec/kb) at 68° C.
3
Molecular Cloning Reagents Sourcing
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High-purity-grade reagents from Merck (Munich, Germany), Sigma (OOO Sigma-Aldrich Rus, Moscow, Russia), and Helicon (Moscow, Russia) were used. Kits for DNA extraction, restriction, and ligation, oligonucleotides, and Taq polymerase were purchased from Evrogen (Moscow, Russia) and Thermo Fisher Scientific RU (Moscow, Khimki, Russia); kanamycin was produced by Sintez (Moscow, Russia). Yeast extract, bactoagar, tryptone, and peptone were purchased from “Helicon” and “Dia-M” (Moscow, Russia). DNA and protein molecular weight markers were purchased from BioRad (Hercules, CA, USA).
4
RNA Extraction and RT-PCR for Virus Detection
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RNA was extracted using TRI Reagent LS (Sigma-Aldrich, St. Louis, MA, USA), according to the manufacturer’s manual. Reverse transcription with random hexamer primers (R6) and MMLV RT kit (Evrogen, Moscow, Russia) was performed on an isolated matrix following the manufacturer’s instructions [15 (link)]. PCR was performed using virus-specific oligonucleotides with Evrogen Taq-polymerase (Evrogen, Moscow, Russia) following the manufacturer’s instructions.
5
Quantifying Gene Expression in Cell Lines
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Total RNA was isolated from hiBC line No 3 on the fourth passage and from primary BCs on the second passage using the RNeasy kit (QIAGEN, Netherlands). cDNA was synthesized from 500 ng of total RNA using MMLV Reverse Transcriptase (Evrogen, Russia) and a mix of oligo (dT) and random primers, according to the manufacturer’s protocol. The housekeeping gene B2M was used as a positive control for the reaction. Then, 2 μL of cDNA was added to each 25 μL end-point PCR. PCR was performed using the gene-specific primers (Table 2 ), Taq polymerase (Evrogen, Russia), and the ProFlex PCR System (Applied Biosystems, USA). The cycling parameters were as follows: 95°C for 5 min, followed by 40 cycles of 20 s at 95°C, 5 s at 58°C, and 5 s at 72°C. The DNA electrophoresis of PCR products was performed in a 2% agarose gel and TBE buffer using Gel Loading Dye Blue (Evrogen, Russia) and a 100-bp + DNA ladder (Evrogen, Russia).
6
Rapid Molecular Diagnostic Kit Development
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Kits for RPA were manufactured by TwisDx (Maidenhead, UK). Biotin- and fluorescein (FAM)- labeled oligonucleotides were synthesized by Syntol (Moscow, Russia). Proteinase K, a mix for PCR containing SYBR Green I, dNTP, Taq polymerase, and a DNA purification kit were purchased from Evrogen (Moscow, Russia). The nitrocellulose membrane CNPC12, PT R5 fiberglass membrane, sample pad membrane GFB-R4, and absorbent pad AP045 were produced by Advanced Microdevices (Ambala Cantt, India). Mouse monoclonal IgG specific to fluorescein (anti-FAM) was produced by Bialexa (Russia). Recombinant streptavidin and goat anti-mouse IgG were produced by Imtek (Russia). HAuCl4, bovine serum albumin (BSA), and lyophilized salmon-sperm DNA were purchased from Sigma-Aldrich (St. Louis, MO, USA). Salts, buffers, organic solvents, and other low-molecular-weight organic compounds were of analytical grade and purchased from different commercial retailers.
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