Herculase 2 fusion polymerase

Manufactured by Agilent Technologies

Sourced in United States, Germany

Herculase II fusion polymerase is a high-fidelity DNA polymerase designed for accurate DNA amplification. It combines Pfu DNA polymerase and a proofreading domain for efficient and reliable DNA synthesis.

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Lab products found in correlation

Qiaquick pcr purification kit, by Qiagen (2 mentions) Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions) Ampure xp bead, by Beckman Coulter (1 mentions) Facsaria 2 sorp, by BD (1 mentions) Dynabeads human t activator anti cd3 28, by Thermo Fisher Scientific (1 mentions) Foxp3 staining solutions, by Thermo Fisher Scientific (1 mentions) Tri reagent, by Merck Group (1 mentions) Zero blunt topo, by Thermo Fisher Scientific (1 mentions) Accuprime taq hifi, by Thermo Fisher Scientific (1 mentions) Quick gdna miniprep kit, by Zymo Research (1 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions) Qiaamp dna mini kit, by Qiagen (1 mentions) Surveyor mutation detection kit, by Integrated DNA Technologies (1 mentions) Kpni bamhi, by New England Biolabs (1 mentions) Kapa sybr fast, by Merck Group (1 mentions) Tapestation d1000, by Agilent Technologies (1 mentions) S220 focused ultrasonicator, by Beckman Coulter (1 mentions) Ampure xp spri beads, by Beckman Coulter (1 mentions) Gentra puregene kit, by Qiagen (1 mentions) Bioruptor pico sonication system, by Diagenode (1 mentions)

21 protocols using herculase 2 fusion polymerase

Optimized PCR Amplicon Libraries for Deep Sequencing

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PCR amplicon libraries for deep sequencing were prepared using an adapted protocol ^{22 (link),46}. All primer sequences are provided in Supplementary Table 2. The first round of PCR (PCR I) was performed on DNA from unsorted cells using 6.6μg of gDNA/100μL PCR reaction. To maintain 300X coverage, 20 reactions were assembled/sample. For sorted cells, all extracted gDNA per sample was distributed into two 100μL reactions. 18-20 cycles of amplification were performed using Herculase II Fusion polymerase (Agilent). The reactions from PCR I were pooled together and used as a template for the second round of PCR (PCR II), to add the necessary adapters for Illumina sequencing. To adjust for varying PCR efficiency between samples, the cycle number for PCR II was adjusted such that each library was amplified in a 50μL reaction to generate ~50ng of DNA library. The final DNA for sequencing was purified using AMPure XP beads (Agencourt) according to manufacturer’s instructions with the following modifications: Each 50 uL PCR II reaction was mixed with 25uL of beads and incubated for 5m. Magnetic separation was used to collect the supernatant. The supernatant was mixed with 90μL beads and incubated for 5m. The supernatant was discarded and beads were washed twice with 200μL of 70% ethanol. The beads were dried for 10-15m. Bound DNA was eluted from the beads using 40μL water.

Suresh S., Chen B., Zhu J., Golden R.J., Lu C., Evers B.M., Novaresi N., Smith B., Zhan X., Schmid V., Jun S., Karacz C.M., Peyton M., Zhong L., Wen Z., Sathe A.A., Xing C., Behrens C., Wistuba I.I., Xiao G., Xie Y., Fu Y.X., Minna J.D., Mendell J.T, & O’Donnell K.A. (2020). eIF5B drives integrated stress response-dependent translation of PD-L1 in lung cancer. Nature cancer, 1(5), 533-545.

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FOXP3 Expression Analysis in Edited CD4+ T Cells

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FOXP3 mRNA expression was detected by RT-PCR in FOXP3^FL edited CD4⁺ T cells and controls after 3 days of reactivation with Human T-Activator anti-CD3/28 Dynabeads (Life Technologies, 1:25 bead:cell ratio). RNA was extracted with TRI Reagent (Sigma-Aldrich), and polyA⁺ mRNA was reverse transcribed into cDNA using SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific). PCR amplification of FOXP3 cDNA was performed using Herculase II fusion polymerase (Agilent Technologies), and primers are listed in table S1. For assessing FOXP3 protein expression by flow cytometry, cells were fixed and permeabilized using FOXP3 staining solutions (eBioscience) and stained with anti-FOXP3 mAb (clone 259D/C7) conjugated to either AF647 (BD Biosciences) or AF488 (BioLegend) following the manufacturer’s instructions. Fluorescence was detected on a FACSAria II SORP (BD Biosciences), analyzed using FlowJo software v4 10.5.0, and median florescence intensity (MFI) was recorded.

Goodwin M., Lee E., Lakshmanan U., Shipp S., Froessl L., Barzaghi F., Passerini L., Narula M., Sheikali A., Lee C.M., Bao G., Bauer C.S., Miller H.K., Garcia-Lloret M., Butte M.J., Bertaina A., Shah A., Pavel-Dinu M., Hendel A., Porteus M., Roncarolo M.G, & Bacchetta R. (2020). CRISPR-based gene editing enables FOXP3 gene repair in IPEX patient cells. Science Advances, 6(19), eaaz0571.

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Targeted CRISPR Genome Editing in Mouse Models of Duchenne Muscular Dystrophy

Cited 2 times

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hDMD del45 mdx and hDMD del45 mdxD2 mice were electroporated as described [12 (link)] with 20µg of px333 plasmid DNA (Addgene 64073, Andrea Ventura [13 (link)]) containing CRISPR guides 44C4 and 55C3 (from [11 (link)]) or pmaxGFP as a control forhDMDdel45 mdxD2mice. In brief, 5µl hyaluronidase was injected into the flexor digitorum brevis (FDB) muscle and 1hr later the DNA was injected and electroporated 20 times for 20 ms at 1 Hz.
hDMD del45 mdx mice were harvested 22 or 33 days later and genomic DNA was extracted by digesting the muscles with proteinase K then using the Quick-gDNA™ Miniprep Kit (Zymo Research). PCR for an exon 45–55 deletion was performed as described using Accuprime Taq HiFi (Thermo Fisher Scientific) or Herculase II Fusion Polymerase (Agilent Genomics) [11 (link)]. Sequencing of blunt cloned PCR products from Zero Blunt^® TOPO^® (Life Technologies) was done by Laragen Inc.
hDMD del45 mdxD2 mice were harvested 24 days post-electroporation. The interosseous (IO) and FDB were flash frozen and samples of 10µm cryosections taken throughout the whole muscle. Intervening sections as well as the lumbricalis were used for genomic DNA extraction and PCR as above.
Please see expanded Materials and Methods in the Supplementary Data.

Young C.S., Mokhonova E., Quinonez M., Pyle A.D, & Spencer M.J. (2017). Creation of a Novel Humanized Dystrophic Mouse Model of Duchenne Muscular Dystrophy and Application of a CRISPR/Cas9 Gene Editing Therapy. Journal of neuromuscular diseases, 4(2), 139-145.

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Analysis of Off-Target Effects in NSMCE2 Null Cells

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Genomic DNA was extracted from WT and NSMCE2 null U2OS cells, respectively. To gain an overview of off-target effects in the established NSMCE2 null cell line, the 10 top-ranking potential off-target sites provided by the online CRISPR Design Tool (http://crispr.mit.edu) were analyzed. The selected sites included those preceding 5′-NAG, the alternative PAM. The genomic regions flanking each potential off-target sites were amplified by PCR with Herculase II fusion polymerase (Agilent Technologies). The selected off-target sites and the corresponding PCR primers are depicted in Table S1. Purified PCR products were Sanger-sequenced for analysis of off-target effects.

Verver D.E., Zheng Y., Speijer D., Hoebe R., Dekker H.L., Repping S., Stap J, & Hamer G. (2016). Non-SMC Element 2 (NSMCE2) of the SMC5/6 Complex Helps to Resolve Topological Stress. International Journal of Molecular Sciences, 17(11), 1782.

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Targeted Sequencing of Ancient Sheep DNA

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In order to increase coverage, we enriched chosen libraries for 20,000 SNPs (see below) via in-solution capture hybridization using custom designed⁵³ MyBaits probes (Arbor Biosciences Inc.) (see Supplementary Data 3 for the SNP list). We used four libraries with >1% endogenous sheep DNA (TEP03, TEP62, TEP83, ULU031) and one library with <1% endogenous sheep DNA (ULU26) (Supplementary Data 2). The biotinylated RNA capture probes were produced by Arbor Biosciences Inc. and capture experiments were implemented following the manufacturer’s instructions. Specifically, hybridization reactions were incubated at 55 °C for 24 h and the captured libraries were amplified using Herculase II Fusion Polymerase (Agilent Technologies) for 15–19 cycles. Enriched libraries were purified using AMPure XP beads and then quantified by Agilent 2100 Bioanalyzer. Purified libraries were pooled in equimolar concentrations to reach a final concentration of 10 nM and then were sequenced paired-end with a 2 × 150 setup using HiSeq X SBS chemistry. We note that ULU26 coverage was only 0.003× after the second round of sequencing and data from this sample was not included in population genetic analyses using nuclear data, but its mitochondrial DNA was used in haplogroup assignment.

Yurtman E., Özer O., Yüncü E., Dağtaş N.D., Koptekin D., Çakan Y.G., Özkan M., Akbaba A., Kaptan D., Atağ G., Vural K.B., Gündem C.Y., Martin L., Kılınç G.M., Ghalichi A., Açan S.C., Yaka R., Sağlıcan E., Lagerholm V.K., Krzewińska M., Günther T., Morell Miranda P., Pişkin E., Şevketoğlu M., Bilgin C.C., Atakuman Ç., Erdal Y.S., Sürer E., Altınışık N.E., Lenstra J.A., Yorulmaz S., Abazari M.F., Hoseinzadeh J., Baird D., Bıçakçı E., Çevik Ö., Gerritsen F., Özbal R., Götherström A., Somel M., Togan İ, & Özer F. (2021). Archaeogenetic analysis of Neolithic sheep from Anatolia suggests a complex demographic history since domestication. Communications Biology, 4, 1279.

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Quantifying CRISPR-Induced Genomic Modifications

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Genomic DNA of transfected U2OS cells was extracted with QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), according to the protocol provided by the manufacturer. The genomic region (417 bp) containing both mutation sites in exon 3 of NSMCE2 was amplified by PCR with Herculase II fusion polymerase (Agilent Technologies, Santa Clara, CA, USA). The forward and reverse primers used for PCR were 5′-AATTTCAAGATGCCAGGACGT-3′ and 5’-GGATCTTCAAATCTTTGCCCAT-3′, respectively. PCR products were purified by QIAquick PCR Purification Kit (Qiagen). Genomic modifications in the amplified region were then detected with Surveyor Mutation Detection Kit (Integrated DNA Technologies, Coralville, IA, USA), according to the manufacturer’s instructions. After Surveyor nuclease digestion, the PCR products were run on a 2% agarose gel with ethidium bromide (EB) for visualization. The insertion/deletion (indel) occurrence was estimated with the formula described by Ran et al. [63 (link)].

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Targeted Disruption of terthp in D. discoideum

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Targeted disruption of the terthp gene in D. discoideum was accomplished using the pLPBLP vector, Cre-loxP system based, with resistance to blasticidin [38 (link)]. Targeting arms were amplified from D. discoideum (AX4) genomic DNA using the Herculase II fusion polymerase (Agilent technologies. CA, USA). The 5′ targeting arm was amplified using the primers: terthp Forward, arm-1 and Reverse, arm-1 (Table 1) which incorporated the KpnI and HindIII target sites to facilitate directional cloning into the vector. It covered the 313 bp upstream to the +1 and the first 736 bp of the gene. The 3′ targeting arm was amplified using the primers: terthp Forward, arm-2 and Reverse, arm-2 (Table 1) which incorporated PstI and BamHI sites. The 3´ arm covered the last 699 pb of the gene plus the next 303 pb. AX4 cells were electroporated with 10 μg of KpnI/BamHI (New England Biolabs. MA, USA) digested vector. Colonies resistant to blasticidin were collected. Genomic DNA was extracted using MasterAmp Buccal Swab DNA Extraction Solution (Epicentre. WI, USA). Gene disruption was validated by PCR reactions with two pair of primers shown in Table 1: 1) terthp Forward, test-1 and Reverse, test-1; 2) nola4 Forward and Reverse (see results)

Rodriguez-Centeno J., Manguán-García C., Perona R, & Sastre L. (2019). Structure of Dictyostelium discoideum telomeres. Analysis of possible replication mechanisms. PLoS ONE, 14(9), e0222909.

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Optimized Illumina Library Preparation from 3C Samples

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3C samples and controls were quantified using Qubit (Invitrogen), run on a 1% agarose gel and tested by qPCR with KAPA SYBR Fast (Sigma) to determine library quality. qPCR primers are in Supplementary Data 1. Only libraries with a digestion efficiency >70% were used for Capture-C. Libraries were either indexed with NEBNext DNA Library Prep Master Mix for Illumina (New England Biolabs) using 6 µg input 3C DNA as previously described following manusfacture’s instructions or using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs). When using the Ultra II kit 3 µg 3C material was sonicated to 200 bp using a Covaris S220 Focused Ultrasonicator, and purified using Ampure XP SPRI beads (Beckman Coulter). DNA was eluted into 53 µL with 1 µL used for D1000 TapeStation analysis (Agilent) and 2 µL used for Qubit quantification (Invitrogen). Fifty microliters of DNA ( ≤2 µg) was then indexed with the following modifications; for the End Prep reaction, the 20 °C incubation was lengthened to 45 min, 5 µL of NEBNext Adaptor was added and incubated for 30 min at 20 °C, the USER Enzyme incubation was extended to 30 min (37 °C), and indexing was performed in two reactions with Herculase II Fusion Polymerase (Agilent) using six cycles of amplification.

Downes D.J., Beagrie R.A., Gosden M.E., Telenius J., Carpenter S.J., Nussbaum L., De Ornellas S., Sergeant M., Eijsbouts C.Q., Schwessinger R., Kerry J., Roberts N., Shivalingam A., El-Sagheer A., Oudelaar A.M., Brown T., Buckle V.J., Davies J.O, & Hughes J.R. (2021). High-resolution targeted 3C interrogation of cis-regulatory element organization at genome-wide scale. Nature Communications, 12, 531.

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Quantifying CRISPR Knockout Efficiency

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To confirm efficient CRISPR cutting at target loci, PCR primers were
designed flanking the sgRNA cut site by 75–100 base pairs on either side.
Human genomic DNA PCR primer sequences corresponding to the regions targeted by
MDR1, SLC26A2, and MTF1 sgRNAs are shown in Supplementary Table 13. Genomic DNA
was isolated from knockout cell lines using the Gentra Puregene Kit (QIAGEN) and
amplified with the primers to yield an amplicon roughly 150–250 base
pairs in length using Herculase II Fusion Polymerase (Agilent). The PCR protocol
involved a 2-minute denaturation at 95C, followed by 24 cycles of 95°C
for 2 minutes, 55°C for 2 min, and 72°C for 1 min. PCR samples
were purified and submitted for the NGS CRISPR sequencing assay at the
Massachusetts General Hospital DNA Core. Knockout efficiency was assessed by the
percentage of reads containing a frameshift caused by an indel compared to the
total read count. Results for each guide were averaged across primer sets with
successful amplification.

Corsello S.M., Nagari R.T., Spangler R.D., Rossen J., Kocak M., Bryan J.G., Humeidi R., Peck D., Wu X., Tang A.A., Wang V.M., Bender S.A., Lemire E., Narayan R., Montgomery P., Ben-David U., Garvie C.W., Chen Y., Rees M.G., Lyons N.J., McFarland J.M., Wong B.T., Wang L., Dumont N., O’Hearn P.J., Stefan E., Doench J.G., Harrington C.N., Greulich H., Meyerson M., Vazquez F., Subramanian A., Roth J.A., Bittker J.A., Boehm J.S., Mader C.C., Tsherniak A, & Golub T.R. (2020). Discovering the anti-cancer potential of non-oncology drugs by systematic viability profiling. Nature cancer, 1(2), 235-248.

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cfDNA Library Preparation Protocol

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The cfDNA was subjected to library preparation as previously described [7 (link)] with modifications. In brief, initially blunt ending and 5′ phosphorylation was performed using T4 polymerase and T4 kinase respectively. Sequencing adaptors were then ligated at both ends using T4 Ligase (New England Biolabs, Ipswich, UK). Nicks were removed in a fill-in reaction using Bst polymerase (New England Biolabs). Library amplification was performed using Herculase II Fusion Polymerase (Agilent Technologies, Santa Clara, CA), and unique barcodes were assigned to all samples. At each step, products were purified using Ampure XP magnetic beads according to manufacturer’s instructions.
Prior to library construction of buccal swab, amniotic fluid and CVS samples, extracted genomic DNA was sheared to an average size of 250 bp using the Bioruptor Pico sonication system (Diagenode, Liege, Belgium). Blunt ending, adaptor ligation and adaptor fill-in reactions were performed without intermediate purification steps. A single purification step was performed following library amplification using Ampure XP magnetic beads according to manufacturer’s instructions.
All library preparation steps were run on Hamilton STAR (Hamilton, Bonaduz, Switzerland) or epMotion (Eppendorf) systems using in-house developed automated methods.

Koumbaris G., Achilleos A., Nicolaou M., Loizides C., Tsangaras K., Kypri E., Mina P., Sismani C., Velissariou V., Christopoulou G., Constantoulakis P., Manolakos E., Papoulidis I., Stambouli D., Ioannides M, & Patsalis P. (2019). Targeted capture enrichment followed by NGS: development and validation of a single comprehensive NIPT for chromosomal aneuploidies, microdeletion syndromes and monogenic diseases. Molecular Cytogenetics, 12, 48.

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