Random prime labeling kit

Manufactured by Roche

The Random Prime Labeling Kit is a laboratory tool used for the labeling of DNA or RNA sequences. It enables the incorporation of labeled nucleotides into DNA or RNA samples through a random priming process. The core function of this kit is to facilitate the efficient and effective labeling of genetic material for various downstream applications, such as hybridization experiments and detection techniques.

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Lab products found in correlation

Redivue α 32p dctp, by Cytiva (1 mentions) Biomax ms film, by Kodak (1 mentions) Turboblotter, by GE Healthcare (1 mentions) Typhoon scanner, by GE Healthcare (1 mentions) Dneasy blood and tissue kit, by Qiagen (1 mentions) Biomax ms, by Kodak (1 mentions)

3 protocols using random prime labeling kit

Cytokine Detection and Immune Response Analysis

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Example 20

Materials and Experimental Methods

Detection of Cytokines in Plasma.

Serum samples obtained from blood collected at different times during and after 7 daily lipofectin-complexed mRNA administrations are analyzed for mouse IFN-α, TNF-α, and IL-12 using ELISA kits.

Northern Blot Analysis.

Aliquots (2.0 μg) of RNA samples isolated from spleen are separated by denaturing 1.4% agarose gel electrophoresis, transferred to charged membranes (Schleicher and Schuell) and hybridized in MiracleHyb® (Stratagene). Membranes are probed for TNF-α, down-stream IFN signaling molecules (e.g. IRF7, IL-12 p35 and p40, and GAPDH) and other markers of immune activation. Specificity of all probes is confirmed by sequencing. To probe the membranes, 50 ng of DNA is labeled using Redivue[α-³²P] dCTP® (Amersham) with a random prime labeling kit (Roche). Hybridized membranes are exposed to Kodak BioMax MS film using an MS intensifier screen at −70° C.

Histopathology.

Spleens from EPO-ψmRNA-treated and positive and negative control-treated mice are harvested, fixed, sectioned, stained with hematoxylin and eosin and examined by a veterinary pathologist for signs of immune activation.

US11801314B2. RNA containing modified nucleosides and methods of use thereof (2023-10-31). The Trustees of the University of Pennsylvania [US]. Inventors: Katalin Kariko [US], Drew Weissman [US].

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HPV Genome Detection and Quantification

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Total DNA was isolated from CIN612-9E cells using the DNeasy blood and tissue kit (Qiagen). DNA (2 μg) was digested with either a single-cut linearizing enzyme (HindIII) for the HPV genome or with a noncutter (BamHI) to linearize cellular DNA. After digestion, samples were separated on a 0.8% agarose-Tris-acetate-EDTA (TAE) gel and transferred onto nylon membranes using a Turbo Blotter (GE Healthcare). Membranes were UV cross-linked (120 mJ/cm²), dried, and prehybridized in hybridization buffer (3× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 2% SDS, 5× Denhardt’s solution, 0.2 mg/ml sonicated salmon sperm DNA) for 1 h. The membrane was hybridized overnight with 25 ng (³²P)-dCTP-labeled HPV31 DNA probe in hybridization buffer. The membrane was washed in 0.1% SDS/0.1× SSC, and hybridized DNA was visualized and quantitated by phosphor-imaging on a Typhoon scanner (GE Healthcare). The ³²P-radiolabeled probe was generated from a plasmid containing the entire HPV16 genome by radiolabeling using a Random Prime labeling kit (Roche).

Khurana S., Markowitz T.E., Kabat J, & McBride A.A. (2021). Spatial and Functional Organization of Human Papillomavirus Replication Foci in the Productive Stage of Infection. mBio, 12(6), e02684-21.

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RCMV RNA Expression Profiling

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RFL6 fibroblasts plated on 10cm dishes were infected with RCMV (MOI=1). At 8, 24 and 48 hpi the cells were washed and lysed with Trizol for 5 min at room temperature. Subsequently, the samples were scraped and stored at −80°C. RNA was isolated per the manufacturer’s instructions and electrophoresed through a 1% agarose/formaldehyde gel and transferred to GeneScreen Plus nylon membranes (Dupont/NEN). The blots were hybridized with probes specific for R116 and GAPDH generated from 500bp BamHI fragments of plasmids containing R116 or GAPDH using Roche Random Prime Labeling kit. Alternatively, single stranded probes were made by end labeling DNA oligonucleotides complementary for R116 or GAPDH sequences using T4 polynucleotide kinase (New England Bio). The Northern blots were hybridized in Express Hybe (Clontech) and washed with low stringency wash (2xSSC with 0.05% SDS) followed by high stringency wash (0.1xSSC with 0.1% SDS). The blots were exposed to autoradiography film (Kodak Biomax MS) using intensifying screens at −80°C, developed, and visualized.

Gatault P., Jones I.K., Meyer C., Kreklywich C., Alexander T., Smith P.P., Denton M., Powell J., Orloff S.L, & Streblow D.N. (2021). Rat and Human Cytomegalovirus ORF116 Encodes a Virion Envelope Glycoprotein Required for Infectivity. Virology, 557, 23-33.

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