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Pfuultra hotstart dna polymerase

Manufactured by Agilent Technologies
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PfuUltra Hotstart DNA Polymerase is a thermostable DNA polymerase designed for high-fidelity DNA amplification. It exhibits robust performance and enhanced specificity for targeted DNA sequences.

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Lab products found in correlation

Ni nta resin, by Thermo Fisher Scientific (3 mentions) β mercaptoethanol β me, by Thermo Fisher Scientific (3 mentions) E coli xl 10 cells, by Agilent Technologies (3 mentions) Deoxynucleotide mix pcr grade, by Agilent Technologies (3 mentions) Acetonitrile hplc grade, by Agilent Technologies (3 mentions) Bovine serum albumin (bsa), by Merck Group (3 mentions) Lb broth, by Merck Group (3 mentions) Quikchange 2 site directed mutagenesis kit, by Agilent Technologies (3 mentions) Thiamine pyrophosphate, by Merck Group (2 mentions) 1 deoxy d xylulose 5 phosphate sodium salt, by Merck Group (2 mentions) T4 ligase, by Thermo Fisher Scientific (1 mentions) Restriction enzyme, by Thermo Fisher Scientific (1 mentions) Hydrogen peroxide, by Merck Group (1 mentions) Lysozyme from chicken egg white, by Merck Group (1 mentions) Cumene hydroperoxide, by Merck Group (1 mentions) Geneclean spin kit, by MP Biomedicals (1 mentions) Epoch spectrophotometer, by Agilent Technologies (1 mentions) Quikchange 2 xl site directed mutagenesis protocol, by Agilent Technologies (1 mentions) Pyruvate, by Merck Group (1 mentions) Lr clonase 2 enzyme mix, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

PfuUltra (22 citations) PfuUltra II Fusion HotStart DNA Polymerase (12 citations) PfuUltra HotStart (3 citations)

9 protocols using pfuultra hotstart dna polymerase

1

Site-Directed Mutagenesis and Enzymatic Assays

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Restriction enzymes and T4 ligase were purchased from Thermo Fisher Scientific (Waltham, MA, United States). Primers used in the site-directed mutagenesis were purchased from Genomed S.A. (Warszawa, Poland). β-casein from bovine milk and lysozyme from chicken egg white, hydrogen peroxide, cumene hydroperoxide, as well as all antibiotics and other chemicals were purchased from Merck (Poznań, Poland). Pfu Ultra hotstart DNA polymerase was from Agilent Technologies (La Jolla, CA, United States).
Zarzecka U., Modrak-Wójcik A., Figaj D., Apanowicz M., Lesner A., Bzowska A., Lipinska B., Zawilak-Pawlik A., Backert S, & Skorko-Glonek J. (2019). Properties of the HtrA Protease From Bacterium Helicobacter pylori Whose Activity Is Indispensable for Growth Under Stress Conditions. Frontiers in Microbiology, 10, 961.
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2

KRAS PCR Amplification from Lung Samples

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High-fidelity, first-round PCR reactions were performed using 1 µg of digested normal lung or lung tumor genomic DNA as template. Each 200 µl PCR reaction contained: 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl (pH 8.75), 2 mM MgSO4, 0.1% Triton X-100, 0.1 mg/ml bovine serum albumin, 0.2 mM dNTPs, 0.2 µM RD1 (5′-TTAAGCGTCGATGGAGGAGTT-3′), 0.2 µM RD2 (5′-GTCCTGCACCAGTAATATGC-3′) and four units of cloned PfuUltra Hotstart DNA Polymerase (Agilent Technologies, CA, USA). The first-round PCR included a 2 min denaturation at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min at 56°C and 1 min at 72°C and a final 7 min extension at 72°C. The 384 bp KRAS PCR product (which included sequence 5′ of exon 1, exon 1 and part of intron 1) was isolated following preparative agarose gel electrophoresis using a Geneclean Spin Kit (MP Biomedicals, Solon, OH), eluted with TE buffer and frozen as multiple single-use aliquots. The concentration of each DNA sample was determined by repeated measurement using an Epoch Spectrophotometer (BioTek, Winooski, VT). Final concentrations were calculated from three determinations that varied by ≤10% from the group mean.
Myers M.B., McKim K.L., Meng F, & Parsons B.L. (2015). Low-frequency KRAS mutations are prevalent in lung adenocarcinomas. Personalized medicine, 12(2), 83-98.
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3

Pfu Ultra Hot Start DNA Amplification

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PCR using the proofreading Pfu ultra hot start DNA polymerase (Agilent Technologies, Santa Clara, CA, USA) was performed according to the manufacturer’s instructions.
Gheit T., Dutta S., Oliver J., Robitaille A., Hampras S., Combes J.D., McKay-Chopin S., Le Calvez-Kelm F., Fenske N., Cherpelis B., Giuliano A.R., Franceschi S., McKay J., Rollison D.E, & Tommasino M. (2017). Isolation and characterization of a novel putative human polyomavirus. Virology, 506, 45-54.
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4

Evaluating NL4.3 Virus Replication

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To test the replication capacity of NL4.3 viruses expressing wild-type HIV-1JR-FL Env or the adapted virus Envs, we introduced mutations into the HIV-1NL4.3(JR-FL) proviral DNA and transfected them into target cells. Site-directed mutagenesis was performed using the QuikChange II XL Site-Directed Mutagenesis protocol (Agilent) and the PfuUltra Hotstart DNA Polymerase (Agilent). Approximately 4×104 Cf2Th-CD4/CCR5 or R5-Low cells preincubated with 50 or 100 ng/mL doxycycline were transfected with 100 μg proviral DNA and passaged for 3 weeks. Supernatants of each cell sample were collected regularly throughout the culture period and evaluated for RT activity.
Espy N., Pacheco B, & Sodroski J. (2017). Adaptation of HIV-1 to Cells with Low Expression of the CCR5 Coreceptor. Virology, 508, 90-107.
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5

Recombinant Enzyme Production Protocol

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Thiamine pyrophosphate (TPP), pyruvate, D,L-glyceraldehyde-3-phosphate (GAP), 1-deoxy-D-xylulose-5-phosphate sodium salt, bovine serum albumin, and LB-broth were purchased from Sigma Aldrich. NADPH was purchased from Alexis Biochemical, Ni-NTA resin was purchased from Invitrogen, and β-mercaptoethanol (β-Me) was purchased from Fisher. E. coli XL-10 cells, deoxynucleotide mix PCR grade, pfuUltra Hotstart DNA polymerase, QuikChange II site directed mutagenesis kit and acetonitrile (HPLC grade) were purchased from Agilent. The DNA vectors, pET28a (+) and pET15b (+), and E. coli BL-21 B (DE3) cells were purchased from EMD Biosciences. DNA sequencing services and primers were purchased from MWG operon. All the other reagents were of the highest quality commercially available.
Handa S., Dempsey D.R., Ramamoorthy D., Cook N., Guida W.C., Spradling T.J., White J.K., Woodcock H.L, & Merkler D.J. (2018). Mechanistic Studies of 1-Deoxy-D-Xylulose-5-Phosphate Synthase from Deinococcus radiodurans. Biochemistry & molecular biology journal, 4(1), 2.
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6

Multilocus Sequence Typing of Cryptococcus neoformans

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DNA was isolated by standard methods from the C. neoformans strains indicated. PCR amplification of multilocus sequence typing (MLST) allelic regions for the highly conserved regions of six genes (CAP59, GDP1, LAC1, PLB1, SOD1, and URA5) was performed with PfuUltra HotStart DNA polymerase (Agilent Technologies) according to the C. neoformans MLST consortium (28 (link)). The primer sequences, consensus MLST loci, and lengths of the trimmed sequence files used were from the Pathogenic Fungi MLST Database (http://mlst.mycologylab.org/defaultinfo.aspx?Page=MLSTconsensustable). PCR products were run on a 2% agarose gel to confirm product size prior to DNA sequencing. Trimmed MLST sequences were concatenated, resulting in the inclusion of 3,271 to 3,463 characters for alignment and phylogenetic analysis with ClustalW (63 (link), 64 (link)). Distances included for the unrooted tree were calculated by using ClustalW phylogeny. Distance values show the number of substitutions as a proportion of the length of the alignment, excluding gaps.
Hu G., Chen S.H., Qiu J., Bennett J.E., Myers T.G, & Williamson P.R. (2014). Microevolution During Serial Mouse Passage Demonstrates FRE3 as a Virulence Adaptation Gene in Cryptococcus neoformans. mBio, 5(2), e00941-14.
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7

Purification and Characterization of Recombinant Enzymes

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TDP, pyruvate, G3P, DXP sodium salt, bovine serum albumin, and LB broth were purchased from Sigma-Aldrich. NADPH was purchased from Alexis Biochemical, Ni-NTA resin was purchased from Invitrogen, and β-mercaptoethanol (β-Me) was purchased from Fisher. E. coli XL-10 cells, deoxynucleotide mix PCR grade, pfuUltra Hotstart DNA polymerase, QuikChange II site directed mutagenesis kit, and acetonitrile (HPLC grade) were purchased from Agilent. The DNA vectors pET28a(+) and pET15b(+) and E. coli BL-21 B(DE3) cells were purchased from EMD Biosciences. DNA sequencing services and primers were purchased from MWG operon. All the other reagents were of the highest quality commercially available.
White J.K., Handa S., Vankayala S.L., Merkler D.J, & Woodcock H.L. (2016). Thiamin Diphosphate Activation in 1-Deoxy-D-xylulose 5-Phosphate Synthase: Insights into the Mechanism and Underlying Intermolecular Interactions. The journal of physical chemistry. B, 120(37), 9922-9934.
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8

Recombinant Enzyme Production Protocol

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Thiamine pyrophosphate (TPP), pyruvate, D,L-glyceraldehyde-3-phosphate (GAP), 1-deoxy-D-xylulose-5-phosphate sodium salt, bovine serum albumin, and LB-broth were purchased from Sigma Aldrich. NADPH was purchased from Alexis Biochemical, Ni-NTA resin was purchased from Invitrogen, and β-mercaptoethanol (β-Me) was purchased from Fisher. E. coli XL-10 cells, deoxynucleotide mix PCR grade, pfuUltra Hotstart DNA polymerase, QuikChange II site directed mutagenesis kit and acetonitrile (HPLC grade) were purchased from Agilent. The DNA vectors, pET28a (+) and pET15b (+), and E. coli BL-21 B (DE3) cells were purchased from EMD Biosciences. DNA sequencing services and primers were purchased from MWG operon. All the other reagents were of the highest quality commercially available.
Handa S., Dempsey D.R., Ramamoorthy D., Cook N., Guida W.C., Spradling T.J., White J.K., Woodcock H.L, & Merkler D.J. (2018). Mechanistic Studies of 1-Deoxy-D-Xylulose-5-Phosphate Synthase from Deinococcus radiodurans. Biochemistry & molecular biology journal, 4(1), 2.
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9

Plasmid DNA Preparation and DNA Assembly

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Plasmid DNA was prepared using the QIAprep Spin Miniprep Kit (QIAGEN) and Econospin columns (Epoch Life Science) according to manufacturer's protocols. PCR reactions were performed with Taq polymerase, PfuUltra Hotstart DNA Polymerase (Agilent Technologies) and Expand High Fidelity PCR System (Roche Life Science) according to manuacturer's protocols. PCR products were purified by Zymoclean Gel DNA Recovery Kit (Zymo Research). All DNA constructs were confirmed through DNA sequencing by Elim Biopharmaceuticals Inc. Restriction enzymes (NEB) and T4 ligase (NEB) were used to digest and ligate the DNA fragments, respectively. BP Clonase II Enzyme Mix, Gateway pDONR221 Vector and LR Clonase II Enzyme Mix (Life Technologies) and the S. cerevisiae Advanced Gateway Destination Vector Kit (Addgene)67 (link) were used to perform Gateway Cloning. Gibson one-pot, isothermal DNA assembly68 (link) was conducted at 10 μl scale by incubating T5 exonuclease (NEB), Phusion polymerase (NEB), Taq ligase (NEB) and 50 ng of each DNA fragment at 50 °C for 1 h to assemble multiple DNA fragments into one circular plasmid. Yeast strains are constructed through homologous recombination and DNA assembly23 (link). Yeast strains and plasmids utilized in this study are listed in Supplementary Data 1 and 2. DNA sequences of genes involved in this work are listed in Supplementary Data 3.
Li Y, & Smolke C.D. (2016). Engineering biosynthesis of the anticancer alkaloid noscapine in yeast. Nature Communications, 7, 12137.
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