Kapa2g robust pcr kit

Manufactured by Roche

Sourced in United States

The KAPA2G Robust PCR Kit is a polymerase-based reagent designed for versatile and robust PCR amplification. The kit includes a proprietary DNA polymerase, optimized buffer, and other necessary components to facilitate efficient DNA amplification.

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Lab products found in correlation

Kingfisher flex purification system, by Thermo Fisher Scientific (3 mentions) Magmax dna multi sample ultra kit, by Thermo Fisher Scientific (3 mentions) Takara la taq kit, by Takara Bio (2 mentions) Mutation surveyor dna variant analysis software, by SoftGenetics (2 mentions) Qscript cdna synthesis kit, by Quanta Biosciences (2 mentions) Rneasy plus mini kit, by Qiagen (2 mentions) Oragene saliva collection kit, by DNA Genotek (1 mentions) Qiaamp dna micro kit, by Qiagen (1 mentions) Amplitaq gold dna polymerase kit, by Thermo Fisher Scientific (1 mentions) Qiaamp dna blood mini kit, by Qiagen (1 mentions) Chromaspro 1, by Technelysium (1 mentions) Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions) Abi prism 3730xl genetic analyzer, by Thermo Fisher Scientific (1 mentions) Qiaquick gel extraction kit, by Qiagen (1 mentions) Bigdye direct cycle sequencing kit, by Thermo Fisher Scientific (1 mentions) Exosap it kit, by GE Healthcare (1 mentions) Dneasy blood tissue kit, by Qiagen (1 mentions) Dneasy blood and tissue kit, by Qiagen (1 mentions) Kapa2g robust dna polymerase, by Roche (1 mentions)

Spelling variants of this product

KAPA2G Robust HotStart PCR Kit (16 citations) KAPA2G Robust HotStart ReadyMix PCR Kit (13 citations) KAPA2G Robust HotStart PCR Ready Mix (2´) kit (2 citations)

19 protocols using kapa2g robust pcr kit

ANKRD11 Quantitative PCR and CNV Analysis

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For PT4 and PT14’s DNA, quantitative PCR assays (qPCR) were carried out using the SYBR green methodology with specific ANKRD11 primer pairs, as previously described [36 (link)]. CNV junction characterization through Long-Range PCR was performed on PT4’s DNA using the TaKaRa LA Taq™ kit (Takara Bio Inc., Shiga, Japan), while the KAPA2G™ Robust PCR Kit (Kapa Biosystems, Wilmington, MA, USA) was used for PT14’s DNA. Amplicons were sequenced by direct dideoxy sequencing and then analyzed, as previously described [36 (link)]. Primers used for CNV characterization are listed in Table S5.

Bestetti I., Crippa M., Sironi A., Tumiatti F., Masciadri M., Smeland M.F., Naik S., Murch O., Bonati M.T., Spano A., Cattaneo E., Mariani M., Gotta F., Crosti F., Cavalli P., Pantaleoni C., Natacci F., Bedeschi M.F., Milani D., Maitz S., Selicorni A., Spaccini L., Peron A., Russo S., Larizza L., Low K, & Finelli P. (2022). Expanding the Molecular Spectrum of ANKRD11 Gene Defects in 33 Patients with a Clinical Presentation of KBG Syndrome. International Journal of Molecular Sciences, 23(11), 5912.

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Genetic Variant Screening in Saliva

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Saliva samples were collected using the Oragene saliva collection kits (DNA Genotek, Inc., Ontario, Canada) from the proband, her affected daughter and her daughter’s father. Genomic DNA was extracted from saliva samples using Oragene Purifier (OG-L2P, DNA Genotek, Inc.) and purified with QIAamp DNA micro kit (#56304, Qiagen, Valencia, CA) according to the manufacturer’s instructions. PCR amplification of the 2 exons of ZNF469 and 16 exons of PRDM5 was performed using previously described primers.^{24 (link), 25 (link)} PCR amplification reactions were performed using the KAPA2G Robust PCR Kit (KAPA Biosystems, Wilmington, MA) with annealing temperature of 65 °C (ZNF469 primers) and 63 °C (PRDM5 primers).^{24 (link), 25 (link)} Purified PCR products were purified using 0.5 U of Shrimp Alkaline Phosphatase and 5 U of Exonuclease I (USB Corp., Cleveland, OH) and underwent Sanger sequencing (Laragen, Inc., Culver City, CA). The resulting sequencing reads were compared to the wild-type ZNF469 transcript (NM_001127464.2) and PRDM5 transcript (NM_018699.3). Minor allele frequencies (MAF) were obtained for each identified variant from Trans-Omics for Precision Medicine (TOPMed) Program, Exome Aggregation Consortium (ExAC) database, 1000 Genomes Project and Genome Aggregation Database (gnomeAD). A variant was determined to be rare if the TOPMed MAF was ≤ 0.01.

Zhang W., Margines J.B., Jacobs D.S., Rabinowitz Y.S., Hanser E.M., Chauhan T., Chung D., Bykhovskaya Y., Gaster R.N, & Aldave A.J. (2019). Corneal perforation following corneal cross-linking in keratoconus associated with potentially pathogenic ZNF469 mutations. Cornea, 38(8), 1033-1039.

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Amplification of Promoter Regions

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Promoter and regulatory regions selected based on the presence of predicted elements by UCSC browser (Tracks: Integrated Regulation from ENCODE → Layered H3K4Me1; ENCODE Histone Modification → Broad ChromHMM) [32 (link)], were amplified using the AmpliTaq Gold DNA Polymerase Kit (Thermo Fisher Scientific) or, in the case of CG-rich regions, the KAPA2G Robust PCR Kit (KAPA BIOSYSTEMS). A full list of primers pairs, and detailed conditions are reported in Supplementary Table S3.

Sironi A., Bestetti I., Masciadri M., Tumiatti F., Crippa M., Pantaleoni C., Russo S., D’Arrigo S., Milani D., Larizza L, & Finelli P. (2022). A unique Smith-Magenis patient with a de novo intragenic deletion on the maternally inherited overexpressed RAI1 allele. European Journal of Human Genetics, 30(11), 1233-1238.

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Extracting and Amplifying DNA for CRISPR Analysis

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Total cell DNA was extracted and purified at 48 hpi using the QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturers instructions. An ~800 bp gene RNA4.9 fragment, covering the target locations of all sgRNAs, was amplified using KAPA 2G Robust PCR kit (KAPABIOSYSTEMS; forward primer AGTGCGCATGCGTCGGTA, reverse primer ACCTACCGTCGTCGTCGG) and used with the Alt-R Genome Editing Detection Kit (IDT) according to the manufacturer’s instructions.

Tai-Schmiedel J., Karniely S., Lau B., Ezra A., Eliyahu E., Nachshon A., Kerr K., Suárez N., Schwartz M., Davison A.J, & Stern-Ginossar N. (2020). Human cytomegalovirus long noncoding RNA4.9 regulates viral DNA replication. PLoS Pathogens, 16(4), e1008390.

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Genotyping ADRB2 Gly16Arg Polymorphism

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The genotype of the ADRB2 Gly16Arg (G/C) polymorphism was examined by extracting and analyzing the DNA from each participant enrolled in the study. In detail, to clarify the genotypes (GG, GC, or CC) of the ADRB2 Gly16Arg (G/C) polymorphism, a buccal swab from each participant was acquired with a sterile cotton swab (Tomy Works, Sakai, Japan). Genotyping was outsourced to EBS (Hiroshima, Japan) as previously described [15 (link)]. Genomic DNA harvested from each subject was extracted from the swab using the KingFisher Flex Purification System (ThermoFisher Scientific, Paisley, UK) and the MagMAX DNA Multi-Sample Ultra Kit (ThermoFisher Scientific) in accordance with the manufacturer’s guidelines. The genotype was determined by polymerase chain reaction (PCR) using the confronting two-pair primer (PCR-CTPP) method and the KAPA2G Robust PCR Kit (Kapa Biosystems, Wilmington, MA, USA) according to the manufacturer’s guidelines.

Narita K., Kudo T.A., Hong G., Tominami K., Izumi S., Hayashi Y, & Nakai J. (2022). Effect of Beta 2-Adrenergic Receptor Gly16Arg Polymorphism on Taste Preferences in Healthy Young Japanese Adults. Nutrients, 14(7), 1430.

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Localization of Deletion Breakpoints

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To localize the deletion breakpoints, long-range PCR was performed on genomic DNA using TaKaRa LA Taq™ kit (TaKaRa, JP) and on cDNA using the KAPA2G Robust PCR Kit (KAPA BIOSYSTEMS, Wilmington, MA, USA) (Primers details are reported in Supplementary Table S1). Amplicons were sequenced using the Big Dye® Terminator v.3.1 Cycle Sequencing kit (Thermo Fisher Scientific). Deletion junction sequences were aligned to the human reference genome sequence (human genome assembly GRCh37/hg19), and electropherograms analyzed with the ChromasPro 1.5 software (Technelysium Pty Ltd., Tewantin QLD, Australia).

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Sanger Sequencing for SNV Validation

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Candidate SNVs were confirmed by Sanger sequencing. Primers were designed using Primer3 (v.4.0.0) online software.^{(23 (link))} Regions of candidate SNVs were amplified using KAPA2G Robust PCR Kit (Kapa Biosystems, Wilmington, MA, USA). PCR products were purified with ExoSAP-IT Kit (GE Healthcare, Aurora, OH, USA) and sequencing reactions were prepared using the BigDye Direct Cycle Sequencing Kit (Life Technologies, Carlsbad, CA, USA). The final products were purified via agarose gel and recovered with the QIAquick Gel Extraction Kit (Qiagen GMBH, Hilden, Germany). Capillary electrophoresis sequencing was performed using an ABI Prism 3730XL Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA). The sequence data were analyzed with Mutation Surveyor DNA Variant Analysis Software (SoftGenetics).

Wang J., Yu T., Wang Z., Ohte S., Yao R.E., Zheng Z., Geng J., Cai H., Ge Y., Li Y., Xu Y., Zhang Q., Gusella J.F., Fu Q., Pregizer S., Rosen V, & Shen Y. (2015). A New Subtype of Multiple Synostoses Syndrome Is Caused by a Mutation in GDF6 That Decreases Its Sensitivity to Noggin and Enhances Its Potency as a BMP Signal. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 31(4), 882-889.

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Determination of ACTN3 Genotype

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The ACTN3 genotype (rs1815739) was determined by collecting oral mucosal samples [26 (link)], and a DNA exercise-exercise gene test kit (Hersires International, Hiroshima, Japan) was used to analyze the samples. We ensured that participants fasted for 30 min prior to the test. Participants were asked to gargle with water and then use a sterile cotton swab (Tomy Works, Sakai, Japan) to rub the inside of their cheek for 1 min. EBS (Hiroshima, Japan) was used to extract DNA from the oral mucosa and determine the ACTN3 genotype. A Mag Max DNA Multi-Sample Ultra Kit (Thermo Fisher Scientific, Paisley, UK) and King Fisher Flex Purification System (Thermo Fisher Scientific were used to extract genomic DNA from the swabs. The ACTN3 genotype (rs1815739) was analyzed by polymerase chain reaction (PCR) with two pairs of primers (PCR-CTPP) using the KAPA2G Robust PCR Kit (Kapa Biosystems, Wilmington, MA, USA). ACTN3 genotypes were classified as XX, RX, and RR.

Taniguchi Y., Makizako H., Nakai Y., Kiuchi Y., Akaida S., Tateishi M., Takenaka T., Kubozono T, & Ohishi M. (2022). Associations of the Alpha-Actinin Three Genotype with Bone and Muscle Mass Loss among Middle-Aged and Older Adults. Journal of Clinical Medicine, 11(20), 6172.

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16S rRNA Gene Amplification for Microbiome Analysis

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PCR reactions were performed in 96-well plates (Fig. 1a). The reaction was designed to amplify the 16S rRNA V3–V9 gene region with unique plate barcodes. The reverse primer contained an overhang with a 9-bp barcode sequence (Supplementary Fig. 1b). To minimize the pipetting variation of small volumes, master mixes were prepared for 100 reactions. The master mix was prepared using the KAPA2G Robust PCR Kit (KK5024; Kapa Biosystems, Wilmington, MA, USA) and contained 400 μL of 5× KAPA2G buffer A, 400 μL of 5× KAPA enhancer, 100 μL of 10 μM forward primer, 100 μL of 10 μM reverse-tagging primer (plate tagging), 40 μL of 10 μM dNTPs, 8 μL of KAPA2G Robust DNA polymerase and 752 μL of sterile deionized water. 18 μL of master mix and 2 μL of DNA template were dispensed into each well of the five 96 well plates. Each plate contained E. coli K-12 MG1655 purified DNA as positive controls. The amplification program was as follows: 5 min denaturation at 95 °C; 25 cycles of 30 s denaturation at 95 °C, 30 s primer annealing at 55 °C, 2 min extension at 72 °C; and a final cooling to 4 °C. The reactions were validated for amplification quality in 1% agarose gel electrophoresis.

Armanhi J.S., de Souza R.S., de Araújo L.M., Okura V.K., Mieczkowski P., Imperial J, & Arruda P. (2016). Multiplex amplicon sequencing for microbe identification in community-based culture collections. Scientific Reports, 6, 29543.

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Quantitative RT-PCR with GAL4 Strains

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Quantitative RT-PCR was conducted as described previously^{20 (link)}. In the experiments using two GAL4 strains with the same selection marker (PTTH-GAL4 and bombyxin-GAL4), genotyping was performed using KAPA2G Robust PCR kit (Kapa Biosystems, MA). Primers used are listed in Supplementary Table S2.

Hara C., Morishita K., Takayanagi-Kiya S., Mikami A., Uchino K., Sakurai T., Kanzaki R., Sezutsu H., Iwami M, & Kiya T. (2017). Refinement of ectopic protein expression through the GAL4/UAS system in Bombyx mori: application to behavioral and developmental studies. Scientific Reports, 7, 11795.

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