We recommend Comparable dynamic range. Wide dynamic range and high sensitivity. Fast primer annealing. Specific primer annealing. Faster results without compromising sensitivity. Resources Supplementary Protocols PDF KB. Kit Handbooks 1. Safety Data Sheets 1. Safety Data Sheets EN. PDF 2MB. Scientific Posters 1.
You will be able to obtain your PCR results in a much shorter time. Do you have any information or guidelines regarding the choice of reference genes for real-time PCR? Do you offer trial-kit sizes for the new QuantiFast Kits? Why do replicates in real-time PCR have different plateau heights? How should fluorescent labeled probes be stored?
What is the threshold cycle or Ct value? The Ct or threshold cycle value is the cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold, a fluorescent signal significantly above the background fluorescence. At the threshold cycle, a detectable amount of amplicon product has been generated during the early exponential phase of the reaction.
The threshold cycle is inversely proportional to the original relative expression level of the gene of interest. How do you achieve fast cycling, yet still deliver the same performance in PCR as that achieved with standard cycling? Why does my realtime PCR assay quality decrease over time? BMC Res Notes. Dec 11; The procedure is fast, easy to automate, and minimizes the risk of contamination due to fewer handling steps.
For further details, please see our online section on ' Critical factors for successful gene expression assays ', or download our Brochure ' Critical Factors for Successful Real-Time PCR '.
See also FAQ for further info. However, 4-plex PCR is possible on on instruments equipped with at least 5 channels. Alternatively, if you have a 4-channel real-time cycler that does not use ROX passive reference dye e. Replicates in real-time PCR may have different plateau heights due to differences in the reaction kinetics for each sample.
Even though replicates start out with identical template amounts, the rate at which reagents are being depleted, and the point when exponential accumulation of PCR product stops and becomes linear, differ between replicates. This will result in different plateau heights, the stage where PCR reactions have come to a halt, and little or no additional PCR product is being amplified.
Fluorescent oligonucleotides should be stored in the dark, as light can slowly degrade the fluorescent moieties.
For optimal long-term storage of fluorescent dye-labeled probes except Cyanine , Cy3. If resuspended below pH 7. Note that Cyanine , Cy3. For best results, resuspend Cy-labeled oligos at pH 7. QuantiTect Kits are also compatible with the Rotor-Gene Q; however, the kits require a significantly longer cycling time.
Such contamination is most often due to the improper disposal of tubes, tips, and gels that previously came into contact with PCR products. Additionally, PCR products may also contaminate pipettors, racks, work pads, and commonly used reagents such as water and buffers.
To minimize the risk of contaminating your experiment with extraneous DNA, the following steps should be taken:. Due to the cloning and purification processes, obtaining recDNA can lengthen the overall process of generating standards. To find a primer assay for your target gene of interest, please visit our GeneGlobe data base. The primers of the QuantiTect Primer Assays are at a proprietary concentration that was specially optimized for sensitive and efficient amplification in any real time cycler.
Excessive exposure to elevated temperatures will result in reactivation of the HotStarTaq Plus DNA Polymerase, eventually leading to nonspecific amplification. Yes, QuantiTect Primer Assays work very well under fast-cycling conditions. The most important prerequisite for any gene expression analysis experiment is the preparation of consistently high-quality RNA from every experimental sample.
Contamination by DNA, protein, polysaccharide, or organic solvents can jeopardize the success of an experiment. Residual polysaccharides, collagen, other macromolecules, and organic solvents in an RNA sample can inhibit the activity of DNase, which may interfere with DNase treatment for genomic DNA removal. These contaminants may also inhibit reverse transcriptase and DNA polymerase, leading to lower reverse transcription efficiency and reduced PCR sensitivity.
For each QuantiTect Primer Assay , we retrieve the sequence of the target gene from curated databases and exclude SNP regions from assay design. We then use our proprietary algorithm to design assays that amplify RNA sequences only i. Successful amplification of the target is indicated by the following factors: high sensitivity, high efficiency, high specificity i.
Depending on the qPCR instrument, time savings when switching from standard cycling e. Generally, 1— ng template should be sufficient and for abundant transcripts as little as 1 pg can be used. Template purity is important if large volumes of low concentration template are to be added to the reaction.
No, QuantiTect Primer Assays are supplied as lyophilized, premixed primer pairs. Shorter amplification products facilitate high PCR efficiencies. Ideally, amplicon length should be less than bp for optimal amplification efficiency. Use of in vitro transcripts as standards takes into account the variable efficiency of the RT reaction. Cell lysates of each subcellular fraction were prepared and immunoprecipitated with either normal mouse IgG, or anti-Ago2 antibodies.
Total protein was isolated from precipitates and resolved by SDS-PAGE and then analysed by western blotting using an Ago2 antibody to validate the efficiency of purification.
Figure Lengend Snippet: The reduced transcriptional activity of the haplotype I Fcgr2b promoter is caused by three single nucleotide substitutions leading to defective AP-1 binding. B The transcriptional activity of the WT promoter mutated at the indicated positions was determined as in A. Article Title: Isolation, identification and expression analysis of salt-induced genes in Suaeda maritima, a natural halophyte, using PCR-based suppression subtractive hybridization.
Actin gene served as the internal control. Each bar represents the number of fold increase in the transcript level of a gene in the plant upon NaCl treatment compared to the control level. Buy from Supplier. If resuspended below pH 7. Note that Cyanine , Cy3. For best results, resuspend Cy-labeled oligos at pH 7.
Due to the cloning and purification processes, obtaining recDNA can lengthen the overall process of generating standards. The most important prerequisite for any gene expression analysis experiment is the preparation of consistently high-quality RNA from every experimental sample.
Contamination by DNA, protein, polysaccharide, or organic solvents can jeopardize the success of an experiment. Residual polysaccharides, collagen, other macromolecules, and organic solvents in an RNA sample can inhibit the activity of DNase, which may interfere with DNase treatment for genomic DNA removal.
These contaminants may also inhibit reverse transcriptase and DNA polymerase, leading to lower reverse transcription efficiency and reduced PCR sensitivity. Shorter amplification products facilitate high PCR efficiencies. Ideally, amplicon length should be less than bp for optimal amplification efficiency. Use of in vitro transcripts as standards takes into account the variable efficiency of the RT reaction.
It is not necessary to perform calibration steps with MAX dye. On the Rotor-Gene Q, use the yellow channel. Reorder now! Reorder from your past orders in just one click. Order by Quote. Quote Number. Add quote number from your quote document. Customer Number. Add customer number from your quote document. To remove a quote go to the Cart. View Quote Example. Catalog Number.
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