Kapa hifi hotstart polymerase

Manufactured by Roche

Sourced in United States

KAPA HiFi HotStart polymerase is a DNA polymerase enzyme used in polymerase chain reaction (PCR) amplification. It exhibits high fidelity and robust performance, enabling accurate and efficient DNA synthesis.

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Lab products found in correlation

Hiseq 2000, by Illumina (13 mentions) Sodium periodate, by Merck Group (4 mentions) T4 rna ligase, by Thermo Fisher Scientific (4 mentions) Rnase h, by Thermo Fisher Scientific (4 mentions) Dna clean concentrator 5, by Zymo Research (4 mentions) Bioruptor, by Diagenode (4 mentions) Dynabeads m 270 streptavidin beads, by Thermo Fisher Scientific (4 mentions) Maxima h minus reverse transcriptase, by Thermo Fisher Scientific (4 mentions) Superscript 3, by Thermo Fisher Scientific (3 mentions) Superscript double stranded cdna synthesis kit, by Thermo Fisher Scientific (3 mentions) Dynabeads mrna purification kit, by Thermo Fisher Scientific (3 mentions) Agencourt ampure xp bead, by Beckman Coulter (3 mentions) Hiseq 4000, by Illumina (3 mentions) Mybaits target enrichment kit, by New England Biolabs (3 mentions) Phusion polymerase, by New England Biolabs (3 mentions) Truseq nano dna library prep kit, by Illumina (2 mentions) T4 rna ligase 2, by New England Biolabs (2 mentions) T4 rna ligase 1, by New England Biolabs (2 mentions) Nextera xt index kit, by Illumina (2 mentions) Nucleospin gel and pcr clean up mini kit, by Macherey-Nagel (2 mentions)

Close spelling variants of this product

KAPA HiFi polymerase (98 citations) KAPA HiFi HotStart DNA polymerase (52 citations) KAPA HiFi (30 citations) KAPA HiFi HotStart (27 citations) HiFi polymerase (17 citations) KAPA HiFi HotStart Uracil+ DNA polymerase (12 citations) KAPA HiFi HotStart PCR polymerase (6 citations) Hotstart polymerase (3 citations) KAPA HiFi HotStart DNA Polymerase with 2X Master Mix (3 citations) KAPA HiFi HotStart ReadyMix DNA Polymerase (3 citations)

43 protocols using kapa hifi hotstart polymerase

3' RNA-Specific Library Preparation

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To obtain 3′ RNA-specific libraries, we essentially used the above-described Tn5 loading and tagmentation protocol with the following changes: undiluted Tn5 stock was loaded exclusively with the Tn5ME-B/Tn5MErev linker. Subsequent PCR enrichment was performed using KAPA HiFi HotStart polymerase (Kapa Biosystems) supplemented with TMAC, i7 adapter index primer, and a custom PE1.Smart-seq2 primer. Importantly, we skipped the gap-filling step in the PCR program to prevent amplification of tagmented cDNA from the gene-coding region.

Hennig B.P., Velten L., Racke I., Tu C.S., Thoms M., Rybin V., Besir H., Remans K, & Steinmetz L.M. (2017). Large-Scale Low-Cost NGS Library Preparation Using a Robust Tn5 Purification and Tagmentation Protocol. G3: Genes|Genomes|Genetics, 8(1), 79-89.

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Degradome-seq Library Construction Protocol

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Degradome-seq library construction was performed as described previously^{45 (link)}. The RNAs were first oxidized at room temperature for 30 min with sodium periodate (Sigma, St. Louis, MO, USA) to block the 3′-ends from ligation and were then size-selected to isolate RNA ≥200 nts (DNA Clean & Concentrator™-5, Zymo Research). 5′ Adapters were attached using T4 RNA ligase (Ambion) at 20 °C for 3 h. The ligated products were subjected to rRNA depletion with complementary DNA oligomers (IDT) and RNase H (Invitrogen)^{140 (link),141 (link)}. The rRNA-depleted ligation products were reverse transcribed using a degenerate primer (Supplementary Table 1, Degenerate primer). cDNA was amplified by PCR using KAPA HIFI Hotstart polymerase (Kapa Biosystems, Wilmington, MA, USA), and 250–350 nts double-stranded DNA was isolated on 8% native PAGE gels.

Sun Y.H., Wang R.H., Du K., Zhu J., Zheng J., Xie L.H., Pereira A.A., Zhang C., Ricci E.P, & Li X.Z. (2021). Coupled protein synthesis and ribosome-guided piRNA processing on mRNAs. Nature Communications, 12, 5970.

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Degradome-seq Library Construction Protocol

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Degradome-seq library construction was performed as described previously^{54 (link)} with modification. The RNAs were first oxidized at room temperature for 30 minutes with sodium periodate (Sigma, St. Louis, MO, USA) to block the 3´-ends from ligation, and were then size-selected to isolate RNA ≥ 200 nt (DNA Clean & Concentrator™-5, Zymo Research). 5´ adaptors were attached using T4 RNA ligase (Ambion) at 20°C for 3 hours. The ligated products were subjected to rRNA depletion with complementary DNA oligomers (IDT) and RNaseH (invitrogen)^{53 (link),55 (link)}. The rRNA depleted ligation products were reverse transcribed using a degenerate primer (5´-GCACCCGAGAATTCCANNNNNNNNC-3´). cDNA was amplified by PCR using KAPA HIFI Hotstart polymerase (Kapa Biosystems, Wilmington, MA, USA), and 250–350 nt dsDNA was isolated on 8% native PAGE gels. An Illumina HiSeq 2000 was used to perform single-end sequencing.

Sun Y.H., Zhu J., Xie L.H., Li Z., Meduri R., Zhu X., Song C., Chen C., Ricci E.P., Weng Z, & Li X.Z. (2020). Ribosomes guide pachytene piRNA formation on long intergenic piRNA precursors. Nature cell biology, 22(2), 200-212.

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PCR Amplification and Next-Gen Sequencing of sgRNA Libraries

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The purified plasmids were used as templates for PCR to amplify the N20 region of the genome-wide library sgRNAs (50 μl × 4 reactions per library; 50 ng template per reaction; PF/R_pTargetLacNGS_PE150 primers; KAPA HiFi HotStart polymerase (KAPA Biosystems); 95°C 3 min, 20 cycles [98°C, 20 s; 67.5°C, 15 s; 72°C, 30 s], 72°C for 1 min). PCR conditions for tiling library is 50 μl × 4 reactions per library, 50 ng template per reaction, PF/R_pTargetLacNGS_SE50, Q5 polymerase, (NEB), 98°C 30 s, 17 cycles [98°C 10 s, 53°C 30 s, 72°C 10 s], 72°C 1 min. The sequencing library was prepared following the manufacturer's protocol (TruSeq DNA Nano Library Prep Kit for Illumina). Sequencing for the genome-wide sgRNA library was carried out using a 2 × 150 paired-end configuration and ∼30 million reads were collected for each library with targeting sgRNAs and 3 million reads for negative control sgRNA libraries (Supplementary Table S4). Illumina NextSeq 500 by the SE50 technique was applied for tiling sgRNA library sequencing.

Guo J., Wang T., Guan C., Liu B., Luo C., Xie Z., Zhang C, & Xing X.H. (2018). Improved sgRNA design in bacteria via genome-wide activity profiling. Nucleic Acids Research, 46(14), 7052-7069.

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Constructing Antibiotic-Resistant Plasmid Mutants

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Mutant plasmid constructs were designed with a 1500 bp Km-cassette flanked by 500-bp sequences corresponding to upstream and downstream regions of the target gene. The Km-cassette and flanking regions co-amplified as one unit by PCR. The upstream (A and B primers) and downstream (C and D primers) flanking regions were PCR amplified (Kapa HiFi HotStart Polymerase, Kapa Biosystems) from A1552 chromosomal DNA and cloned into pJET1.2 for sequencing and amplification purposes. Correct pJET1.2 constructs were digested with XhoI and XbaI and the desired fragments were gel purified. The Km-cassette was PCR amplified from plasmid pKD4 (FRTup and FRTdo primers, DreamTaq Green PCR Master Mix (Fermenta)) and gel-purified. Fifty to 90 ng of the flanking region DNA and 200 ng of the Km-cassette DNA were used for cross-over PCR (Kapa HiFi HotStart Polymerase, Kapa Bisystems) with the following conditions: anneal 43 °C–20 s, elongation 69 °C–1.45 min). The reaction was purified on a gel where the resulting 2500-bp fragments were excised and cloned into pJET1.2 for sequencing and amplification purposes. Correct constructs were digested with NruI and the gel-purified fragments were cloned into SmaI-digested pCVD442 yielding plasmids pCVD-190,5::Km, pCVD-2478,5::Km, and pCVD-526,5::Km.

Sjöström A.E., Sandblad L., Uhlin B.E, & Wai S.N. (2015). Membrane vesicle-mediated release of bacterial RNA. Scientific Reports, 5, 15329.

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Plasmid Extraction and DNA Purification Protocol

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Plasmid extraction and DNA purification were performed using kits from Omega Bio-Tek. Restriction enzyme FastDigest Eco31I (namely BsaI) was purchased from Thermo Scientific. PCR reactions were carried out using KAPA HiFi HotStart polymerase from KAPA Biosystems (NGS library preparation) and Q5 High-Fidelity DNA polymerase from New England Biolabs (cloning). Plasmids were constructed through Gibson assembly. Antibiotic concentrations for kanamycin, ampicillin and chloramphenicol were 50, 100 and 7 mg/l, respectively. All strains and plasmids used in this work are listed in Supplementary Table S1. All oligonucleotides were ordered from Taihe Biotechnology and Genewiz (Supplementary Table S2).

Feng H., Guo J., Wang T., Zhang C, & Xing X.H. (2021). Guide-target mismatch effects on dCas9–sgRNA binding activity in living bacterial cells. Nucleic Acids Research, 49(3), 1263-1277.

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Small RNA Library Preparation Protocol

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For preparation of small RNA libraries, 5 μg RNA (small RNA fraction) was size-selected (<40 nt) by polyacrylamide gel electrophoresis (FlashPAGE, Life Technologies) and precipitated. About 30 ng small RNA (<40 nt) was successive ligated (T4 RNA Ligase 1 and T4 RNA Ligase 2, NEB) to modified 3′ and 5′ adapters (TrueQuant RNA adapters, GenXPro). Adapter-ligated RNA was reverse transcribed (SuperScript III, Life Technologies) and amplified by PCR (KAPA HiFi Hot-Start Polymerase, KAPA Biosystems). Amplified libraries were size-selected by polyacrylamide gel electrophoresis (PAGE) and sequenced (HiSeq2000, Illumina).

Müller S., Raulefs S., Bruns P., Afonso-Grunz F., Plötner A., Thermann R., Jäger C., Schlitter A.M., Kong B., Regel I., Roth W.K., Rotter B., Hoffmeier K., Kahl G., Koch I., Theis F.J., Kleeff J., Winter P, & Michalski C.W. (2015). Next-generation sequencing reveals novel differentially regulated mRNAs, lncRNAs, miRNAs, sdRNAs and a piRNA in pancreatic cancer. Molecular Cancer, 14, 94.

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Single-Cell RNA-seq Library Preparation

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Cells were dissociated using trypsin and resuspended in 100 µl of RNAprotect Cell Reagent (Qiagen) per 100,000 cells. Directly prior to FACS sorting, the cell suspension was diluted with PBS (Gibco). Single cells were sorted into 96-well DNA LoBind plates (Eppendorf) containing lysis buffer using a Sony SH800 sorter (Sony Biotechnology; 100 µm chip) in “Single Cell (3 Drops)” purity. Lysis buffer consisted of a 1:500 dilution of Phusion HF buffer (New England Biolabs). After sorting, plates were spun down and frozen at −80 °C. Libraries were prepared as previously described^{6 (link),8}. Briefly, proteins were digested with Proteinase K (Ambion) followed by desiccation to inactivate Proteinase K and reduce the reaction volume. RNA was then reverse transcribed in a 2 µl reaction at 42 °C for 90 min. Unincorporated barcode primers were digested using Exonuclease I (Thermo Fisher). cDNA was pooled using the Clean & Concentrator-5 kit (Zymo Research) and PCR amplified with the KAPA HiFi HotStart polymerase (KAPA Biosystems) in 50 µl reaction volumes.

Bagnoli J.W., Ziegenhain C., Janjic A., Wange L.E., Vieth B., Parekh S., Geuder J., Hellmann I, & Enard W. (2018). Sensitive and powerful single-cell RNA sequencing using mcSCRB-seq. Nature Communications, 9, 2937.

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Small RNA Sequencing of Serum Samples

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For RNA sequencing, RNA was isolated using the ExoRNeasy Midi kit with a starting volume of 100 µL serum (see above). sncRNA libraries were prepared using TrueQuant technology (GenXPro, Frankfurt am Main, Germany) for elimination of PCR bias. Briefly, modified 3′ and 5′ TrueQuant adapters were successively ligated to small RNA (<200 nt) using T4 RNA Ligase 2 and T4 RNA Ligase 1 (NEB, Frankfurt am Main, Germany), respectively. Adapter-ligated RNA was reverse transcribed with SuperScript III (Life Technologies, Darmstadt, Germany) and amplified by PCR with KAPA HiFi Hot-Start Polymerase (KAPA Biosystems, Wilmington, DE, USA). Amplified libraries were sequenced with HiSeq2000 (Illumina, San Diego, CA, USA). The data provided in the present publication have been deposited in NCBI’s Gene Expression Omnibus [22 (link)] and are accessible through GEO Series accession number GSE138107: (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE138107).

Lipps C., Northe P., Figueiredo R., Rohde M., Brahmer A., Krämer-Albers E.M., Liebetrau C., Wiedenroth C.B., Mayer E., Kriechbaum S.D., Dörr O., Nef H., Hamm C.W., Keller T, & Troidl C. (2019). Non-Invasive Approach for Evaluation of Pulmonary Hypertension Using Extracellular Vesicle-Associated Small Non-Coding RNA. Biomolecules, 9(11), 666.

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Pm4 Gene Amplification Protocol

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The Whealbi collection was screened for the presence of the Pm4 locus using the Pm4 haplotype-specific marker JS717×JS718. Given the difficulty of amplifying the full-length genomic fragment of Pm4 due to the presence of a 4.5 kb intron between exons 5 and 6 that greatly reduced PCR efficiency, we decided to amplify the gene in two parts. The first part corresponds to the genomic region spanning exons 1 to 5 and the second part to exons 6 to 7. To amplify exons 1 to 5, a long range PCR was performed using the primers JS256×JS257 followed by a nested PCR with JS251xJS257. PCR amplification was done using KAPA Hifi HotStart Polymerase (KK2502, Kapa Biosystems) following manufacturer’s recommendations and with an annealing temperature of 60°C and extension time of 2:00 min. The PCR products were sequenced with the internal primers GH382, GH384, GH385 and JS255. For the amplification of the second part of the gene, a long range PCR using the primers JS278xJS261 followed by a nested PCR with JS278xGH407 was done similarly to the PCR dedicated to amplify the first part of the gene but with an annealing temperature of 63°C and an extension time of 3:00. The PCR products were sequenced with the internal primers JS280, JS292, GH387, GH397 and GH402.

Sánchez-Martín J., Widrig V., Herren G., Wicker T., Zbinden H., Gronnier J., Spörri L., Praz C.R., Heuberger M., Kolodziej M.C., Isaksson J., Steuernagel B., Karfiátová M., Doležel J., Zipfel C, & Keller B. (2021). Wheat Pm4 resistance to powdery mildew is controlled by alternative splice variants encoding chimeric proteins. Nature plants, 7(3), 327-341.

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