Hotstar hifidelity polymerase kit

Manufactured by Qiagen

Sourced in Germany

The HotStar HiFidelity Polymerase Kit is a high-fidelity DNA polymerase designed for PCR amplification. The enzyme exhibits proofreading activity, which helps to reduce the rate of mistakes during DNA synthesis.

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Lab products found in correlation

33 protocols using hotstar hifidelity polymerase kit

Cloning and Tagging of CD44 Isoforms

Cited 13 times

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Human CD44v3–10 open reading frame (ORF) was obtained from the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. CD44s cDNA was amplified using a HotStar HiFidelity Polymerase Kit (Qiagen Inc., Hilden, Germany) using LN229 cDNA as a template. The CD44s and CD44v3–10 ORFs were subcloned into a pCAG-Ble-ssPA16 vector possessing signal sequence and N-terminal PA16 tag (GLEGGVAMPGAEDDVV) [28 (link),34 (link),35 (link),36 (link),37 (link)], which is detected by NZ-1, which was originally developed as an anti-human podoplanin mAb [38 (link),39 (link),40 (link),41 (link),42 (link),43 (link),44 (link),45 (link),46 (link),47 (link),48 (link),49 (link),50 (link),51 (link),52 (link),53 (link)].
CHO/CD44s and CHO/CD44v3–10 were established by transfecting the plasmids into CHO-K1 cells using a Neon transfection system (Thermo Fisher Scientific, Inc.). CD44ec-secreting LN229 (LN229/CD44ec) was established by transfecting pCAG-Neo/PA-CD44ec-RAP-MAP into LN229 cells using the Neon transfection system. The amino acid sequences of the tag system in this study were as follows: PA tag [43 (link),47 (link),51 (link)], 12 amino acids (GVAMPGAEDDVV); RAP tag [54 (link),55 (link)], 12 amino acids (DMVNPGLEDRIE); and MAP tag [56 (link),57 (link)], 12 amino acids (GDGMVPPGIEDK).

Suzuki H., Ozawa K., Tanaka T., Kaneko M.K, & Kato Y. (2023). Development of a Novel Anti-CD44 Variant 7/8 Monoclonal Antibody, C44Mab-34, for Multiple Applications against Oral Carcinomas. Biomedicines, 11(4), 1099.

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IGHV1-2 Locus Amplification and Sequencing

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The IGHV1-2 locus was PCR amplified from genomic DNA (25 ng) of each donor examined using the Qiagen HotStar HiFidelity Polymerase Kit (Catalog No. 202602), with previously published oligos (5’-GAGACTCTGTCACAAACAAACCA-3’; 5’-GTGTGTTCTCTTTCTCATCTTGGA-3’). Thermocycler conditions included an initial incubation at 95 °C for 5 min, followed by 30 cycles of: 94 °C for 15 s, 60 °C for 1 min, 72 °C for 1 min, and final extension at 72 °C for 10 min. The resulting PCR product was cloned using the TOPO™ TA Cloning™ Kit, with One Shot™ TOP10 Chemically Competent E. coli (Catalog Number K4575J10). Briefly, TOPO cloning reactions were prepared for each PCR product using the manufacturer’s protocol. Five colonies were selected for Mini-Prep (Catalog No. 27104), and extracted DNA was Sanger sequenced using T7 and SP6 oligos. Allele sequences were confirmed by visual inspection of sequence chromatograms (Supplementary Fig. 3).

Lee J.H., Toy L., Kos J.T., Safonova Y., Schief W.R., Havenar-Daughton C., Watson C.T, & Crotty S. (2021). Vaccine genetics of IGHV1-2 VRC01-class broadly neutralizing antibody precursor naïve human B cells. NPJ Vaccines, 6, 113.

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Sanger Sequencing of Influenza A(H1N1)pdm09 HA Genes

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The influenza A(H1N1)pdm09 isolates were collected after growth in MDCK cells and their identify confirmed by using haemagglutination inhibition assay kits, provided by the CDC in 2017. The isolates with HA titre of more than 8 haemagglutinating units were selected for HA genetic analysis. RNA extraction was conducted on a 140 µl aliquot of each isolate using the viral RNA extraction kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instruction. Sanger sequencing was used to determine the nucleotide sequence of the HA gene. Briefly, cDNA was first performed using the influenza A virus universal primer (Uni 12) AGC AAA AGC AGG as described (SuperScript® III First-Strand Synthesis System, Thermo Scientific, MA, USA), followed by PCR with HA specific primer (HotStar HiFidelity Polymerase Kit, Qiagen, Valencia, CA, USA). The PCR products were purified with PCR purification kits (Qiagen, Valencia, CA, USA) and sequenced using Big Dye Terminator v3.1 (Thermo Scientific, MA, USA) on an ABI 3130 automatic DNA sequencer.

Hoang Vu Mai P., Ung Thi Hong T., Nguyen Le Khanh H., Nguyen Thanh T., Le Thi T., Nguyen Vu S., Nguyen Phuong A., Tran Thi Thu H., Vuong Duc C, & Le Quynh M. (2019). Missed detections of influenza A(H1)pdm09 by real-time RT–PCR assay due to haemagglutinin sequence mutation, December 2017 to March 2018, northern Viet Nam. Western Pacific Surveillance and Response Journal : WPSAR, 10(1), 32-38.

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Biotin-Labeled Targeting Plasmid Generation

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Targeting plasmid template○

Refer to supplemental information for further notes on design of targeting plasmid template

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Primer set for template generation (forward and reverse)•

Both primers must contain a biotin modification on the 5′ end.

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High Fidelity Taq Polymerase kit with proof-reading activity (e.g. HotStar HiFidelity Polymerase kit, Qiagen Cat. No. 202602)

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PCR purification system (e.g. QIAquick PCR purification kit, Qiagen Cat. No. 28104)

Pineault K.M., Novoa A., Lozovska A., Wellik D.M, & Mallo M. (2019). Two CRISPR/Cas9-mediated methods for targeting complex insertions, deletions, or replacements in mouse. MethodsX, 6, 2088-2100.

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Biotin-Tagged DNA Template Generation

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•

Targeting plasmid template○

Refer to method validation for further information on design of targeting plasmid template.

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Primer set for template generation (forward and reverse)•

One primer must contain a biotin modification on the 5′ end

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High Fidelity Taq Polymerase kit with proof reading activity (e.g. HotStar HiFidelity Polymerase kit, Qiagen Cat. No. 202602)

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PCR purification system (e.g. QIAquick PCR purification kit, Qiagen Cat. No. 28104)

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Dynabeads MyOne Streptavidin C1 beads (Thermo Fisher Cat. No. 65001)

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Magnetic tube rack

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3 M NaOAc, pH 5.3

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1 M Tris HCl, pH 7.5

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100% ethanol

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Buffer A•

10mM Tris HCl pH 7.5

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30mM NaCl

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1mM EDTA

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Buffer B

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0.15

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1mM EDTA

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Dry ice

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Tris-EDTA (TE) buffer•

10mM Tris HCl

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1mM EDTA

Pineault K.M., Novoa A., Lozovska A., Wellik D.M, & Mallo M. (2019). Two CRISPR/Cas9-mediated methods for targeting complex insertions, deletions, or replacements in mouse. MethodsX, 6, 2088-2100.

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Overexpression and Silencing of AtGRDP2 in Arabidopsis

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AtGRDP2 ORF was amplified from cDNA of 15 day-old Arabidopsis plants with Hot Star HiFidelity Polymerase Kit (Qiagen, Hilden, Germany) using the primers: AtGRDP2-ORF-F and AtGRDP2-ORF-R primers (Table S1). The product of 2377 bp was cloned into the pCR8/GW/TOPO vector (Invitrogen, Carlsbad, CA, USA), and was sequenced using the M13-F and AtGRDP2-ORF-R primers. The entry clone was recombined into the destination vector pMDC32 using the Gateway LR Clonase Enzyme mix (Invitrogen) to generate pMDC32-GRDP2 vector.
To silence the AtGRDP2 gene, an artificial miRNA pAmiR-AtGRDP2 vector from Thermo Fisher Scientific Inc. was acquired (Waltham, MA, USA). This vector contains 27 bp of the AtGRDP2 gene between the miR319a harpin sequence embedded in their genomic context, 35S CaMV promoter, and the BASTA resistance (Schwab et al., 2006 (link)).

Ortega-Amaro M.A., Rodríguez-Hernández A.A., Rodríguez-Kessler M., Hernández-Lucero E., Rosales-Mendoza S., Ibáñez-Salazar A., Delgado-Sánchez P, & Jiménez-Bremont J.F. (2015). Overexpression of AtGRDP2, a novel glycine-rich domain protein, accelerates plant growth and improves stress tolerance. Frontiers in Plant Science, 5, 782.

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Synthetic mRNA Preparation and Modification

Cited 2 times

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The synthetic mRNAs (eGFP, CD39) were prepared as previously described.4 (link),16 (link) The vectors with coding sequences were amplified with a HotStar HiFidelity Polymerase Kit (Qiagen, Germany) using two primers, TTG GAC CCT CGT ACA GAA GCT AAT ACG as the forward primer (Ella Biotech, Germany) and T₁₅₀ CTT CCT ACT CAG GCT TTA TTC AAA GAC CA as the reverse primer (Ella Biotech, Germany). DNA sequences were purified (Qiaquick PCR purification Kit, Qiagen, Germany), and in vitro transcription (IVT) was performed using a MEGAscript® T7 Kit (Ambion, Scotland), according to the manufacturer's instructions. The mRNA was modified using the 3'-0-Me-m⁷G(5') ppp(5')G RNA cap structure analog (New England Biolabs, Germany), pseudouridine-5'-triphosphate (TriLink Biotech, U. S. A.), and 5-methylcytidine-5'-triphosphate (TriLink Biotech, U. S. A.). Additionally, an RNase inhibitor (Themo Scientific, U. S. A.) was added. The IVT was incubated for 4 h at 37 °C.
Afterwards the mRNA was cleaned up with an RNeasy kit (Qiagen, Germany), and eluted in 40 µl nuclease-free water. Next the dephosphorylation of mRNA was implemented with an Antarctic Phosphatase Kit (New England Biolabs, Germany), cleaned up and then eluted in the same amount of nuclease-free water. The concentration of mRNA was confirmed photometrically and the purity of mRNA with 1% agarose gel.

Abraham M.K., Peter K., Michel T., Wendel H.P., Krajewski S, & Wang X. (2017). Nanoliposomes for Safe and Efficient Therapeutic mRNA Delivery: A Step Toward Nanotheranostics in Inflammatory and Cardiovascular Diseases as well as Cancer. Nanotheranostics, 1(2), 154-165.

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Dual-Index Primer Library Preparation

Cited 1 time

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Libraries were prepared using dual-index primers, as previously described (16 (link), 18 (link), 55 (link)). PCR was performed using the Qiagen HotStar HiFidelity polymerase kit. Touchdown PCR was used to amplify the samples with the following steps: 95°C for 5 min, 35 cycles of 95°C for 45 s, 50°C for 1 min, and 72°C for 1 min, and 72°C for 10 min. A negative control was included for each sample to identify contamination, which was identified using a 1% agarose gel. AMPure beads were used to remove the primer dimer, and the Qubit high-sensitivity (HS) assay kit was used to quantify the concentrations. Samples were pooled, gel purified, and sequenced using the Illumina MiSeq machine on a 2 × 250 paired-end run.

Liechty Z., Santos-Medellín C., Edwards J., Nguyen B., Mikhail D., Eason S., Phillips G, & Sundaresan V. (2020). Comparative Analysis of Root Microbiomes of Rice Cultivars with High and Low Methane Emissions Reveals Differences in Abundance of Methanogenic Archaea and Putative Upstream Fermenters. mSystems, 5(1), e00897-19.

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Cloning and Sequencing of ALK, AXL, and GAS6

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The kinase domain of ALK and full-length AXL were amplified from cDNA of SH-SY5Y and SY5Y-TR1 cells using the HotStar HiFidelity Polymerase Kit (Qiagen). The PCR products were cloned into the pGEM®-T vector (Promega) and confirmed by sequencing. The GAS6 insertion (GAS6-SV) was similarly amplified from the two cell lines, and the gel purified PCR product confirmed by sequencing.

Debruyne D.N., Bhatnagar N., Sharma B., Luther W., Moore N.F., Cheung N.K., Gray N.S, & George R.E. (2015). ALK inhibitor resistance in ALKF1174L-driven neuroblastoma is associated with AXL activation and induction of EMT. Oncogene, 35(28), 3681-3691.

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EpCAM Deletion Mutant Expression

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The Genome Network Project clone IRAK021G03 (EpCAM) was provided by the RIKEN BioResource Research Center through the National BioResource Project of the MEXT and AMED agencies of Japan. EpCAM cDNA plus a C-terminal PA tag that is recognized by the anti-PA tag mAb (NZ-1), was subcloned into a pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). N-terminal PA-tagged EpCAM deletion mutants (dN44, dN64, dN84, dN104, dN124, dN144, dN164, dN184, dN204, dN224, and dN244) were produced using a HotStar HiFidelity Polymerase Kit (Qiagen Inc., Hilden, Germany), and subcloned into the pCAG-Ble vector.

Li G., Suzuki H., Asano T., Tanaka T., Suzuki H., Kaneko M.K, & Kato Y. (2022). Development of a Novel Anti-EpCAM Monoclonal Antibody for Various Applications. Antibodies, 11(2), 41.

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