Surestart taq dna polymerase

Manufactured by Agilent Technologies

Sourced in United States

SureStart Taq DNA polymerase is a thermostable DNA polymerase enzyme used in the amplification of DNA sequences during polymerase chain reaction (PCR) experiments. It provides reliable and consistent performance in a variety of PCR applications.

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Lab products found in correlation

Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions) Nanodrop 1000, by Thermo Fisher Scientific (2 mentions) Rnase free dnase 1, by Agilent Technologies (1 mentions) Nuclease free water, by Qiagen (1 mentions) Rnase free dnase 1, by Qiagen (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) 7500 thermocyler, by Thermo Fisher Scientific (1 mentions) Epitect bisulfite kit, by Qiagen (1 mentions) Surecycler 8800 thermal cycler, by Agilent Technologies (1 mentions) Qiaamp dna mini kit, by Qiagen (1 mentions) Qiaquick pcr purification kit, by Qiagen (1 mentions)

5 protocols using surestart taq dna polymerase

RNA Extraction and cDNA Synthesis

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RNA was prepared for each sample using the RNeasy Mini Kit (QIAGEN, Cat. No. 74104, Mississauga, Canada), and genomic DNA was eliminated by loading RNase-free DNase I into the filter column (QIAGEN). Nucleic acid extraction was performed according to the manufacturer's instructions. The quality of the RNA was checked by electrophoresis on 1% agarose gels to reveal any contaminating genomic DNA and intact rRNA subunits 28S and 18S. Nucleic acid concentrations were measured using a Nanodrop 1000 (Thermo Scientific, Wilmington, DE, USA).
First-strand cDNA was synthesized using random hexamer primers and SureStart Taq DNA polymerase according to the manufacturer's instructions (Agilent Technologies, Cat. No.20820, Santa Clara, CA, USA). To avoid errors caused by contamination with genomic DNA, all RNA samples were treated with RNase-free DNase I before reverse transcription (Agilent Technologies, USA). One microgram of total RNA was used for cDNA synthesis; the cDNA was subsequently diluted with nuclease-free water (QIAGEN) to 20 ng/μL. The cDNA mixtures were diluted 1:10 with nuclease-free water (QIAGEN) and stored at −20 °C for subsequent quantitative PCR analysis.

Chen C., Xie T., Ye S., Jensen A.B, & Eilenberg J. (2016). Selection of reference genes for expression analysis in the entomophthoralean fungus Pandora neoaphidis. Brazilian Journal of Microbiology, 47(1), 259-265.

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Quantitative PCR for DNA Extraction Yield

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To ascertain the DNA extraction yields, 5 ul of extracted DNA was amplified via quantitative PCR (qPCR). Using the Surestart Taq DNA polymerase (Agilent, Santa Clara, CA), real-time PCR was performed on an Applied Biosystems 7500 thermocyler under the following conditions: 10 min at 95°C for polymerase activation, followed by 30 cycles of 30 sec at 95°C, 15 sec at 55°C for primer annealing, and 90 sec at 60°C for amplification. The 25 μL reaction mixture contained 1X TaqMan buffer, 3.5mM MgCl2, 8% DMSO, 200uM dNTPs, 200nM primers and TaqMan probes (Table S1), 0.1X Rox Reference Dye, 0.625U Taq DNA polymerase, and 5 μL of sample or standard DNA. For clinical specimen gold standard extraction experiments, a multiplexed HPV 16 and RNaseP qPCR assay was run following the same reaction conditions where RNaseP served as a DNA control to confirm that each clinical specimen did in fact contain cells and that the Qiagen extractions were performed properly. If a clinical sample was negative for RNaseP (cycle threshold value > 30), the sample was deemed invalid and was not used for further experiments.
In each qPCR run, a cycle threshold value versus DNA concentration standard curve was generated from a dilution series of our cloned HPV 16 DNA standards. For each patient sample, the effective viral DNA concentration was quantitated via standard curve interpolations.

Rodriguez N.M., Wong W.S., Liu L., Dewar R, & Klapperich C.M. (2016). A Fully Integrated Paperfluidic Molecular Diagnostic Chip for the Extraction, Amplification, and Detection of Nucleic Acids from Clinical Samples. Lab on a chip, 16(4), 753-763.

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Comprehensive Methylation Analysis of CRP, SAA, and SAP

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Frozen tumor/normal tissue sample pairs were obtained from the tissue bank of Gansu Cancer hospital. Genomic DNA samples extracted from tissues or Hep3B cells were bisulfite-converted and recovered using EpiTect Bisulfite Kit (QIAGEN, Hamburg, Germany; catalog number: 59104; lot number: 142338839, 145038568, 148214306, 151030901) according to the manufacturer’s instructions. Samples were then amplified with SureStart Taq DNA Polymerase (Agilent Technologies, Santa Clara, CA; catalog number: 600282; lot number: 0006129848). The primer sequences used were: human CRP (Forward: 5’-GTAGGTGTTGGAGAGGTAGTTATTA-3’; Reverse: 5’-ATTTATATCCAAAACAATAAAAAAATTTAC-3’); rabbit CRP (Forward: 5’-ATGTTAGAGTTGAAGGTGTTGGAGATA-3’; Reverse: 5’-AAATACTAAAAATCCTACATCCCTTACCTC-3’); human SAA (Forward: 5’-GTTTTTATTTTATATTTTTTAGTAG-3’; Reverse: 5’-TAATACTAATCTATACTATAACTAAACTAC-3’); human SAP (Forward: 5’-AAGAAAGAAAAGGTTTTGTTTTTA-3’; Reverse: 5’-ATTTTCCAAATCTACCTCCTAAC-3’)). Subsequent cloning and sequencing were performed as described (Varley et al., 2009 (link)). The experiments conformed to the Guide for the Care and Use of Laboratory Animals published by NIH, and were conducted according to the protocols approved by the Ethics Committee of Animal Experiments of Xi’an Jiaotong University and Lanzhou University (2016–064 and A201307050027).

Zhang S.C., Wang M.Y., Feng J.R., Chang Y., Ji S.R, & Wu Y. (2020). Reversible promoter methylation determines fluctuating expression of acute phase proteins. eLife, 9, e51317.

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Primer Design and Sequence Validation for Livestock cDNA

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The specificity of each primer pair was checked in preliminary conventional PCR assays with bovine, ovine and caprine cDNA synthesized from Tempus™ total RNA. All PCR reactions were performed with SureStart Taq DNA Polymerase (Agilent Technologies, Inc. Santa Clara, USA) following the manufacturer’s instructions, with 10 ng of cDNA in 0.5 μM of each primer. PCR reactions were conducted on a SureCycler 8800 Thermal Cycler (Agilent Technologies, Santa Clara, USA). The PCR program consisted in an initial denaturation step of 10 min at 95°C, followed by 40 cycles of 30 sec at 95°C, 30 sec at 60°C, and 45 sec at 72°C, followed by a final elongation step of 10 min at 72°C. PCR products were analyzed on 2% agarose gel. Amplicon size was checked with agarose electrophoresis migration (amplicon sizes are listed in Table 2). Finally, PCR products were (i) purified with QIAquick PCR Purification Kit (Qiagen Ltd., Crawley, UK), according to the manufacturer’s instructions, (ii) quantified using a NanoDrop™ ND-1000 Spectrophotometer (Thermo Fisher Scientific, MA, USA), and (iii) sequenced (Beckman Coulter Genomics, Takeley, UK).
The absence of genomic DNA (gDNA) amplification was checked with bovine, ovine and caprine gDNA isolated from blood buffy coat, using the QIAamp DNA Mini Kit (Qiagen Ltd., Crawley, UK).

Puech C., Dedieu L., Chantal I, & Rodrigues V. (2015). Design and evaluation of a unique SYBR Green real-time RT-PCR assay for quantification of five major cytokines in cattle, sheep and goats. BMC Veterinary Research, 11, 65.

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Real-Time PCR for Target Sequence Amplification

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SureStart Taq DNA polymerase (Agilent, Santa Clara, CA) real-time PCR conditions were optimized to include 1 mM magnesium sulphate in the reaction with 5 μl of sample solution in 20 μl of master mix according to the manufacturer’s instructions. Primers and probe, purchased from Integrated DNA Technologies (Coralville, IA) as described in Table 2, were used in the master mix. Samples were heated to 95°C for 10 minutes followed by 45 cycles of 95°C for 15 seconds and 65°C for 45 seconds. Amplification was additionally confirmed via polyacrylamide gel electrophoresis followed by extraction and sequencing of bands. Details of the electrophoresis procedure and results of sequencing are available in the ESI^†.

Linnes J.C., Fan A., Rodriguez N.M., Lemieux B., Kong H, & Klapperich C.M. (2014). Paper-based molecular diagnostic for Chlamydia trachomatis. RSC advances, 4(80), 42245-42251.

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