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发布于:2020-11-27 13:34:49  访问:0 次 回复:0 篇
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30]]["Q Neuroscience"
In this case, two design parameters in the software may be adjusted, amplicon length and amplicon melting temperature. The amplicon length should be a minimum of basepairs, and maximum of basepairs, and if necessary, should be adjusted in basepair increments.Once the primers have been chosen, a general BLAST of each primer sequence is run to ensure their unique specificity.Oligonucleotides are purchased from a commercial vendor at the smallscale synthesis with the minimum purification.We have found that additional purification, such as HPLC, is not necessary and only adds to the cost of each primer.The smallscale synthesis also adds to the savings, especially in the case that the primers do not work initially.To validate the primers, a template titration assay is done.For mouse transcripts, a mixture of equal quantities of RNA from each of the following tissues is used: adrenal, whole brain, embryo, hear t, intestine, kidney, liver, lung, muscle, ovary, pancreas, placenta, macrophage, skin, testis, and white adipose tissue.The assay consists of a fold dilution series of cDNA reversetranscribed from the universal RNA, and a no reverse transcriptase control.Amplification of the NTC sample indicates the presence of primerdimer formed during the reaction.The difference in cycle time between the NTC and the experimental samples must be greater than seven to ensure that the assay is Targetmol‘s Wnt&Hedgehog&Notch Compound Library monitoring specific fluorescence of the experimental sample. The RT sample is included to confirm the absence of genomic amplification.After the template titration assay run is complete, the ABI HT instrument software, SDS.A valid primer set should have a slope of. for the standard curve.Each sample is run in triplicate for each gene to be assayed.Reaction components are premixed in well PCR tubestrips and then pipetted with a multichannel pipette into the reaction plate.Premixing the components decreases the amount of userintroduced variation by ensuring a homogeneous mix of template and primers, and also by minimizing the number of pipettes, which can introduce purchase Anti-Parasite Library bubbles andor aerosol contaminants, into the optical plate itself. The plate is then covered with an optical adhesive cover and centrifuged to bring the liquid to the bottom of the wells of the plate.The instrument software calculates the quantity of transcript in each unknown sample based on the linear regression formula of the standard curve, and data are expor ted as a tabdelimited text file.The relative levels of the mRNA transcript are shown. S rRNA was used as the normalizer gene so that the level of LXR may be compared between tissue types.Outlier points CV are removed from the calculations.The calibrator is chosen to be the sample, the tissue, the gene, or the control group to which the others will be compared.For example, in an experiment in which wildtype mice are compared to knockout mice, the calibrator would be the wildtype mouse sample.The resulting induction values are usually plotted as a bar graph.If there are multiple samples in multiple treatment groups, the average the fold induction for each group is plotted.The protocols and the worksheets provided are intended to introduce and standardize the assay for researchers in the nuclear receptor field, but may be applied to any field of study.
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