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#QFDN-250 QuantiFluo™ DNA Assay Kit
Product name : QuantiFluo™ DNA Assay Kit
Catalog number : QFDN-250
Supplier name : Bioassays
Data sheet: Ask more or other datasheet now !
About this Product :QuantiFluo™ DNA Assay Kit antibody storage GENTAUR recommends for long therm storage to freeze at -24 C. For short time storage up to 30 days we suggest fridge storage at 1 to 10 C. Prevent multiple freeze taw cycles of QuantiFluo™ DNA Assay Kit.
More Details about
For quantitative determination of DNA.
Samples: plasmid DNA, genomic DNA, cDNA, DNA following polymerase chain reaction, and DNA extracted from gel and other matrices.
Procedure: 5 min.
Size: 250 tests.
Detection limit: 100 ng/mL.
DNA quantitation is a common practice in molecular biology. Very often
DNA is available in minute quantities and the traditional UV 260 nm
absorbance method requires microgram quantities for reliable results.
Accurate determination of DNA concentration, especially when DNA is
present at low concentrations, is crucial for reproducible results in
sequencing, cloning, transfection and DNA labeling.
Simple, direct and automation-ready procedures for measuring DNA
concentration are very desirable. BioAssay Systems' QuantiFluoTM DNA
assay kit is designed to accurately measure nanogram quantities of
plasmid DNA, cDNA, DNA following polymerase chain reaction and DNA
eluted from gels. The improved method utilizes Hoechst dye that binds
specifically with double-stranded DNA. The fluorescence intensity,
measured at 450nm (lexc = 350nm), is directly proportional to the DNA
concentration in the sample. The optimized formulation substantially
reduces interference by substances in the raw samples.
Sensitive and accurate. Linear detection range 2 ng to 40 ng (100 –
2,000 ng/mL) calf thymus DNA in 96-well plate assay.
Simple and high-throughput. The “mix-and-read” procedure involves
addition of a single working reagent and reading the fluorescence
intensity. Can be readily automated as a high-throughput assay for
thousands of samples per day.
Low interference. RNA, salt (up to 3M NaCl), detergent (< 0.01% SDS)
and common DNA extraction buffer do not interfere in the assay.
Direct Assays: plasmid DNA, genomic DNA, cDNA, DNA following
polymerase chain reaction, and DNA extracted from gel and other matrices.
KIT CONTENTS (250 tests in 96-well plates)
Reagent: 50 mL Standard: 1 mL 10 μg/mL calf thymus DNA
Storage conditions. The kit is shipped at room temperature. Store the
DNA standard at -20°C and the Reagent at 2-8°C. Shelf life: 12 months
Precautions: reagents are for research use only. Normal precautions for
laboratory reagents should be exercised while using the reagents.
MATERIALS REQUIRED, BUT NOT PROVIDED
Pipeting devices and accessories. 1 x TE buffer.
Procedure using 96-well plate:
Black 96-well plates (e.g. Corning) and fluorescence plate reader.
Procedure using cuvette:
Fluorescence spectrophotometer and fluorometric cuvets
Sample Type: cell line
References: Bhavnani BR et al (2008). Structure activity relationships and differential interactions and functional activity of various equine estrogens mediated via estrogen receptors (ERs) ERalpha and ERbeta. Endocrinology. 149(10):4857-70.
Pubmed ID: 18599548
Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=18599548
Abstract: The human estrogen receptors (ERs) alpha and beta interact with 17beta-estradiol (17beta-E2), estrone, 17alpha-estradiol, and the ring B unsaturated estrogens, equilin, 17beta-dihydroequilin, 17alpha-dihydroequilin, equilenin, 17beta-dihydroequilenin, 17alpha-dihydroequilenin, Delta8-estrone, and Delta8, 17beta-E2 with varying affinities. In comparison to 17beta-E2, the relative binding affinities of most ring B unsaturated estrogens were 2- to 8-fold lower for ERalpha and ERbeta, however, some of these unique estrogens had two to four times greater affinity for ERbeta than ERalpha. The transcriptional activity of these estrogens in HepG2 cells transfected with ERalpha or ERbeta, or both, and the secreted-alkaline phosphatase gene showed that all estrogens were functionally active. 17beta-E2 induced the activity of secreted-alkaline phosphatase by ERalpha to a level higher than any other estrogen. Activity of other estrogens was 12-17% that of 17beta-E2. In contrast, 17beta-E2 stimulated the activity of ERbeta to a 5-fold lower level than that with ERalpha, whereas the activity of other estrogens was 66-290% that of 17beta-E2, with equilenin being the most active. The presence of both ER subtypes did not alter the functional activity of 17beta-E2, although it further enhanced the activity of 17beta-dihydroequilin (200%), 17beta-dihydroequilenin (160%), and Delta8, 17beta-E2 (130%). Except for 17beta-E2, no correlation was observed between the functional activities and their binding affinities for ER. In conclusion, our results show that the effects of ring B unsaturated estrogens are mainly mediated via ERbeta and that the presence of both ER subtypes further enhances their activity. It is now possible to develop hormone replacement therapy using selective ring B unsaturated estrogens for target tissues where ERbeta is the predominant ER.
PMID:18599548 [PubMed - indexed for MEDLINE]
Sample Type: cells, stem cells
References: Mayer-Wagner S et al (2010). Membrane-based cultures generate scaffold-free neocartilage in vitro: influence of growth factors. Tissue Eng Part A. 16(2):513-21.
Pubmed ID: 19715388
Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=19715388
Abstract: Scaffold-free cultures provide promising potential in chondrogenic differentiation of human mesenchymal stem cells (hMSCs). In this study, a novel scaffold-free membrane-based culture system, in which hMSCs were cultivated on a cellulose acetate membrane filter at medium-gas interface, was evaluated for chondrogenesis under the addition of growth factors. Chondrogenic differentiation of hMSCs has been described in scaffold-free pellet cultures with good results. In our study membrane-based cultures (1 x 10(6) hMSCs) were produced, maintained at the medium-gas interface and cultured for 21 days. Results were compared with findings from standard pellet cultures (2.5 x 10(5) hMSCs). The effects of the following growth factors were examined: human transforming growth factor-beta(3) (TGF-beta(3)) +/- insulin-like growth factor-1 or +/- human fibroblast growth factor 2. After 3 weeks of culture, chondrogenesis was assessed by Safranin-O staining, immunohistochemistry, a dimethylmethylene blue dye binding assay for glycosaminoglycans, and quantitative real-time polymerase chain reaction for cartilage-specific proteins. Membrane-based cultures containing growth factors formed hemispherical structures with a large surface area (65 mm(2)). When removed from the membrane they showed a histologically smooth cartilage-like surface. Membrane-based cultures stained positive for Safranin-O and collagen type II and contained a high content of glycosaminoglycans. Expression of cartilage-specific markers like collagen type II, aggrecan, and SOX9 was observed under the addition of TGF-beta(3), whereas combinations of growth factors let to a significant increase of collagen type II expression. A markedly reduced expression of collagen type X was found in membrane-based cultures when only TGF-beta(3) was added. Pellet cultures showed similar results besides an increased expression of collagen type X and type II that were observed. Membrane-based cultures provide a differentiation system, comparable in chondrogenesis to pellet cultures, which is able to generate scaffold-free neocartilage. The key benefit factors of membrane-based cultures are a histologically smooth cartilage-like surface and reduced expression of collagen type X, both of which are suitable features for its future application in cartilage regeneration.
PMID: 19715388 [PubMed - indexed for MEDLINE]
Sample Type: stemcell
References: Mayer-Wagner S et al (2011). Effects of low frequency electromagnetic fields on the chondrogenic differentiation of human mesenchymal stem cells. Bioelectromagnetics. 32(4):283-90.
Pubmed ID: 21452358
Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=21452358
Abstract: Electromagnetic fields (EMF) have been shown to exert beneficial effects on cartilage tissue. Nowadays, differentiated human mesenchymal stem cells (hMSCs) are discussed as an alternative approach for cartilage repair. Therefore, the aim of this study was to examine the impact of EMF on hMSCs during chondrogenic differentiation. HMSCs at cell passages five and six were differentiated in pellet cultures in vitro under the addition of human fibroblast growth factor 2 (FGF-2) and human transforming growth factor-β(3) (TGF-β(3) ). Cultures were exposed to homogeneous sinusoidal extremely low-frequency magnetic fields (5 mT) produced by a solenoid or were kept in a control system. After 3 weeks of culture, chondrogenesis was assessed by toluidine blue and safranin-O staining, immunohistochemistry, quantitative real-time polymerase chain reaction (PCR) for cartilage-specific proteins, and a DMMB dye-binding assay for glycosaminoglycans. Under EMF, hMSCs showed a significant increase in collagen type II expression at passage 6. Aggrecan and SOX9 expression did not change significantly after EMF exposure. Collagen type X expression decreased under electromagnetic stimulation. Pellet cultures at passage 5 that had been treated with EMF provided a higher glycosaminoglycan (GAG)/DNA content than cultures that had not been exposed to EMF. Chondrogenic differentiation of hMSCs may be improved by EMF regarding collagen type II expression and GAG content of cultures. EMF might be a way to stimulate and maintain chondrogenesis of hMSCs and, therefore, provide a new step in regenerative medicine regarding tissue engineering of cartilage.
Copyright © 2010 Wiley-Liss, Inc.
PMID: 21452358 [PubMed - in process]
Sample Type: microglial cells
References: Łabuzek K et al (2010). Metformin has adenosine-monophosphate activated protein kinase (AMPK)-independent effects on LPS-stimulated rat primary microglial cultures. Pharmacol Rep. 62(5):827-48.
Pubmed ID: 21098866
Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=21098866
Abstract: The results of recent studies suggest that metformin, in addition to its efficacy in treating type 2 diabetes, may also have therapeutic potential for the treatment of neuroinflammatory diseases in which reactive microglia play an essential role. However, the molecular mechanisms by which metformin exerts its anti-inflammatory effects remain largely unknown. Adenosine-monophosphate-activated protein kinase (AMPK) activation is the most well-known mechanism of metformin action; however, some of the biological responses to metformin are not limited to AMPK activation but are mediated by AMPK-independent mechanisms. In this paper, we attempted to evaluate the effects of metformin on unstimulated and LPS-activated rat primary microglial cell cultures. The presented evidence supports the conclusion that metformin-activated AMPK participates in regulating the release of TNF-α. Furthermore, the effects of metformin on the release of IL-1β, IL-6, IL-10, TGF-β, NO, and ROS as well as on the expression of arginase I, iNOS, NF-κB p65 and PGC-1α were not AMPK-dependent, because pretreatment of LPS-activated microglia with compound C, a pharmacological inhibitor of AMPK, did not reverse the effect of metformin. Based on the present findings, we propose that the shift of microglia toward alternative activation may underlie the beneficial effects of metformin observed in animal models of neurological disorders.
PMID: 21098866 [PubMed - indexed for MEDLINE]
Sample Type: primary glial culture
References: Labuzek K et al (2010).Ambivalent effects of compound C (dorsomorphin) on inflammatory response in LPS-stimulated rat primary microglial cultures. Naunyn Schmiedebergs Arch Pharmacol. 381(1):41-57.
Pubmed ID: 19940979
Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=19940979
Abstract: It was proven that compound C displays beneficial effects in models of inflammatory-induced anemia, ischemic stroke, and fibrodysplasia ossificans progressiva. Compound C influence on microglia, playing a major role in neuroinflammation, has not been evaluated yet. The aim of the present study was to determine the effect of compound C on cytokine release, NO, and reactive oxygen species (ROS) production. The rat microglial cultures were obtained by shaking the primary mixed glial cultures. Cytokine and nitrite concentrations were assayed using ELISA kits. ROS were assayed with nitroblue tetrazolium chloride. AMPK activity was assayed using the SAMS peptide. The expression of arginase I, NF-kappaB p65, and hypoxia-inducible factor-1 alpha (HIF-1 alpha) was evaluated using Western blot. Compound C displayed ambivalent effect depending on microglia basal activity. It up-regulated the release of TNF alpha and NO production and increased the expression of arginase I in non-stimulated microglia. However, compound C down-regulated IL-1 beta, IL-6 and TNF alpha release, NO, ROS production, and AMPK activity, diminished NF-kappaB and HIF-1 alpha expression, as well as increased arginase I expression in lipopolysaccharide (LPS)-stimulated microglia. Compound C did not affect iNOS expression and IL-10 and TGF-beta release in non-stimulated and LPS-stimulated microglia. The observed alterations in the release or production of inflammatory mediators may be explained by the changes in NF-kappaB, HIF-1 alpha, and arginase I expression and 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolinum bromide values in response to LPS, whereas the basis for the compound C effect on non-stimulated microglia remains to be investigated.
PMID: 19940979 [PubMed - indexed for MEDLINE]
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