Evagreen dye method digital PCR EvaGreen® is a dye with a green excitation wavelength that binds to all dsDNA double helix regions. As a new generation of green fluorescent dye EvaGreen®, it has the following three advantages: 1. The inhibition of PCR is small, so EvaGreen® can use the rapid PCR temperature cycle in the experiment, and can use higher concentration to increase the brightness and eliminate the “dye redistributionâ€; 2. It is extremely stable, will not be destroyed during storage, operation and PCR, and can be freeze-thawed repeatedly; 3. Reduce cell membrane permeability and be safer. The Dye DPCR Experimental Reaction System contains a pair of PCR primers for amplifying specific segments of the sequence of interest and EvaGreen® dyes for amplification products. The steps of the EvaGreen® experiment are as follows 1. Primer and amplicon design The principle of primer design is the same in qPCR and digital PCR. Specific design principles can be found in the previously published primer probe design principles. 2. Experimental preparation For any digital PCR experiment you need to prepare: Digital PCR instrument and special consumables PCR mix contains enzymes, dNTPs, buffers, Mg+ Evagreen dH2O Ordinary PCR laboratory supplies: tube chip oscillator centrifuge, etc. 3. Reaction system Configure MIX as follows: 4. Reaction conditions *: Determined based on the Tm value of the experimental primer 5. Data collection Depending on the system used, data acquisition will take the form of chip imaging (NaicaTM system, QuantStudioTM 3D digital PCR system, CONSTELLATION® digital PCR system...) or similar to flow cytometry (QX200TM droplet digital PCR system) , RaindropTM Digital PCR System), read partitions one by one. Please follow the manufacturer's instructions to perform this step. 6. Data analysis In the EvaGreen digital PCR experiment, only one fluorescence channel needs to be examined, so the data will be represented as a one-dimensional dot plot. Each of the points represents a partition, the y-axis represents the fluorescence intensity of each partition in the blue detection channel, and the x-axis represents the indicator number assigned to each partition by the analysis software. 6.1 Data Quality Control Depending on the type of data collection, data analysis software can provide different quality controls. Two criteria should be carefully considered: NTC: NTC only uses negative droplets Total number of droplets: A large number of analyzable partitions will reduce uncertainty associated with measured concentrations On the NaicaTM system, you can also visualize the generated partitions: 6.2 Threshold setting Typically, the threshold is automatically set by the analysis software, the partition above the threshold is positive, and the partition below the threshold is negative. Therefore, setting the threshold is a mandatory step for digital PCR to process the quantitative results. Since the threshold setting is automatic, we recommend that you view the point one-dimensional map. However, the following two situations require manual threshold setting: â— When there are very few positive partitions or very few negative partitions; â— When you observe a lot of raindrops between the positive and negative partitions. 6.3 concentration calculation After setting the threshold, the software automatically quantifies the number of positive and negative partitions for each target. This digital reading is then converted to a copy number using a Poisson distribution. Each result has a 95% confidence interval that reflects the accuracy of the measurement. The accuracy of the method can also be given by the "95% confidence interval" value, the smaller the better! The 95% confidence interval for each sample concentration and its associated relative uncertainty are automatically calculated by the analysis software. Figure: Evagreen digital PCR experiments have the advantages of simple design, fast establishment and low cost. However, because DNA binding Evagreen is non-specifically binding double-stranded DNA, Evagreen method is mostly used in low-throughput, single-plex experiments. More Evagreen dye methods can be found in the Gene-Ï€ Digital PCR Academy ( Digital PCR Academy Quick Entry) statistical tools, you can also choose other interesting application tutorials, from experimental design to data analysis. Detailed workflow. You can also search for specific items directly by searching the navigation tools ("DNA Preparation for dPCR", "Fluorescence Overflow", etc.), or click on our "HOW TO" to select the part you need (design, analysis, report).
Microscopes provide a maginified view of objects, such as plant elements, microorganisms or tissue. Typically, they are used for objects that are outside of the human visible spectrum. In the microscopes category, we offer you a wide array of high-quality, optical microscopes for your surgery, lab or educational institution. With us, you can buy monocular and binocular microscopes, as well as stereoscopic and digital microscopes.
Combined with microscope, you also will find a wide range of microscope slides and slide covers of the highest lab quality. The compatible accessories for your microscope slides, such as Slides Storage Box and Slides Staining Racks also are available in Yingmed.
Related products like Petri Dishes, Micropette mechanical Pipettes, Pipette tips, Transfer pipettes also will be found in our other category.
Digital Microscope,Microscope Price,Stereo Microscope,Microscope Camera NINGBO YINGMED MEDICAL INSTRUMENTS CO.,LTD , https://www.chinayingmed.com
