Surestart taq polymerase

Manufactured by Agilent Technologies

Sourced in United States, United Kingdom

SureStart Taq polymerase is a thermostable DNA polymerase enzyme used for DNA amplification in PCR (Polymerase Chain Reaction) applications. It provides reliable and consistent DNA amplification by minimizing non-specific amplification and primer-dimer formation.

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Taq polymerase (5 citations) SureStart Taq DNA polymerase (5 citations)

5 protocols using surestart taq polymerase

Optimized qPCR Ligation Product Detection

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25 μL PCR reactions containing 5 μL of ligation product sample were prepared with 0.625 units of SureStart Taq polymerase (Agilent), 4 mM MgCl2, 800μM dNTPs, 100 nM each primer, 250 nM EvaGreen^®, 1.5 nM ROX passive reference dye, and 1X manufacturer-supplied reaction buffer. The MgCl2 concentration reflects the final concentration upon addition of the ligation product template in T4 DNA ligase buffer, which constitutes 2 mM of the final 4 mM concentration. Only two primers were used for PCR amplification of ligation products, namely the common primer and appropriate variable primer. Reactions were heated to 95°C for 10 minutes to activate the hot start polymerase. 40 PCR cycles were performed, each consisting of 95°C for 15 s, 67°C for 30 s, and 72°C for 60 s.

Amaral L.A., Scala A., Barthélémy M, & Stanley H.E. (2000). Classes of small-world networks. Proceedings of the National Academy of Sciences of the United States of America, 97(21), 11149-11152.

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Quantitative PCR for Lambda DNA

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DNA loss and recoveries from each experiment were ascertained via quantitative polymerase chain reaction (qPCR). Samples were amplified using SureStart Taq polymerase (Agilent, Santa Clara, CA, USA) using an Applied Biosystems 7500 thermocycler. Custom primers and Taqman probe sequences were designed for specific sequences of λ-DNA. The forward primer was (5'- GTG GAA TGA ACA ATG GAA GTC AAC AA -3'), the reverse primer was (5'- GGC AGA GTC ATA AAG CAC CTC ATT A -3') (Integrated DNA technologies), and the Taqman probe was (5'- AGG TGC TAC GGC GGC AGA GT -3') tagged with 6-FAM at the 5’-end and a MGB-NFQ quencher at the 3’-end (Applied Biosystems, Waltham, MA, USA). The resulting amplicon was 177 base pairs long. Each 25 μL reaction contained 5 μL of the purified DNA filtrates. Samples were placed in a 96-well plate (Applied Biosystems, Waltham, MA, USA), initially heated to 95°C to activate the polymerase and then cycled 40 times through 30 sec of 95°C for DNA denaturing, 5 sec of 65°C for primer annealing, and 30 sec of 72°C primer extension. qPCR generates curves of the relative fluorescence (ΔRn) after each temperature cycle. The amount of starting DNA is calculated based on the cycle at which the fluorescence in a given well reached a threshold value as shown in Fig 2.

Katevatis C., Fan A, & Klapperich C.M. (2017). Low concentration DNA extraction and recovery using a silica solid phase. PLoS ONE, 12(5), e0176848.

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Quantitative PCR for X. laevis Gene

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25 μL PCR reactions containing 5 μL of extracted nucleic acid were prepared with 0.625 units of SureStart Taq polymerase (Agilent, Santa Clara, CA), 3mM MgCl₂, 800μM dNTPs, 200 μM forward primer, 200 μM reverse primer, and 200 μM TaqMan probe), 1.5 nM ROX passive reference dye, and 1X final concentration of the manufacturer-supplied buffer. Samples were heated to 50°C for 2 minutes followed by 95°C for 10 minutes to activate the polymerase and then 35 PCR cycles were performed, each consisting of 95°C for 15 s, 67°C for 60 s, and 72°C for 60 s. A standard curve was generated using tenfold dilutions of the X. laevis control plasmid from 10⁶ to 10² copies.

Rosenbohm J.M., Robson J.M., Singh R., Lee R., Zhang J.Y., Klapperich C.M., Pollock N.R, & Cabodi M. (2020). Rapid electrostatic DNA enrichment for sensitive detection of Trichomonas vaginalis in clinical urinary samples. Analytical methods : advancing methods and applications, 12(8), 1085-1093.

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DNA Extraction and Quantification

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DNAwas extracted from reconstituted patient samples using a column-based Qiagen Blood and Tissue kit (Qiagen Corporation, Hilden, Germany). Following DNA extraction, DNA integrity was confirmed by detection of the human RNAseP housekeeping gene and NG genomic loads were quantified via SureStart Taq polymerase (Agilent, Santa Clara, CA) qPCR per manufacturer’s instructions on a QuantStudio5 Real-Time PCR system (Thermo Scientific, Waltham, MA). Primers and probes listed in Table S1 were purchased from Integrated DNA Technologies (Coralville, IA). Samples were heated to 95 °C for 10 min followed by 45 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 15 s.

Horst A.L., Rosenbohm J.M., Kolluri N., Hardick J., Gaydos C.A., Cabodi M., Klapperich C.M, & Linnes J.C. (2018). A paperfluidic platform to detect Neisseria gonorrhoeae in clinical samples. Biomedical microdevices, 20(2), 35.

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RT-PCR analysis of GRIN gene expression

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RNA was extracted in Trizol reagent (Thermo Fisher Scientific, Loughborough, UK), any contaminating DNA was digested using a DNA-free kit (Thermo Fisher Scientific), and cDNA was synthesized using Oligo(dT) 12-18 primers (Invitrogen). RT-PCR was performed using SureStart Taq polymerase (Agilent Technologies LDA UK Ltd., Stockport, UK) and a thermal cycler (PCR Express; Hybaid, Thermo Fisher Scientific). With the following annealing temperatures, primers were designed to amplify GRIN1 (forward, 5′-GCATCCTCGGGCTGCAGCTC-3′ and reverse, 5′-AGCGGCCCGGTCTTCCAGAT-3′, 61°C), GRIN2A (forward, 5′-GTGGTCTATCAACGGGCAGT-3′ and reverse, 5′-AGGTGAGACGGTGCCATTAC-3′, 51°C), GRIN2B (forward, 5′-GATGGGAGCCCCTACGCCCA-3′ and reverse, 5′-CCACCGTGGGCTGCCTGAAG-3′, 60°C), GRIN2C (forward, 5′-CGACGCCAGCCACGTGAGTT-3′ and reverse, 5′-AGAGCACCTCGGCCTCCTCG-3′, 54°C, with 5% dimethyl sulfoxide added to the reaction), GRIN2D (forward, 5′-CCACCTTCCTGCAGCTGGGC-3′ and reverse, 5′-GAGCTGGGCACTGAGCACGG-3′, 61°C), glyceraldehyde-3-phosphate dehydrogenase (forward, 5′-ACCCAGAAGACTGTGGATGG-3′ and reverse, 5′-TTCTAGACGGCAGGTCAGGT-3′, 60°C), and UPK2 (forward, 5′-CTCCCGCAAGTAAGGAGGT-3′ and reverse, 5′-GAAGGATGGGGGAATTGTTA-3′, 58°C) transcripts. Reverse transcription negative controls were included in all experiments.

Baker S.C., Shabir S., Georgopoulos N.T, & Southgate J. (2016). Ketamine-Induced Apoptosis in Normal Human Urothelial Cells: A Direct, N-Methyl-d-Aspartate Receptor–Independent Pathway Characterized by Mitochondrial Stress. The American Journal of Pathology, 186(5), 1267-1277.

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