| Description | Inquire | DescriptionUse in combination with the KitAlysis Bench Top Inertion Box (Z742064) or a glove box/glove bag to provide inert atmosphere for kit set-up.Designed to be used with KitAlysis High-Throughput Screening Kits.Components:24-Well Reaction BlockTorque ScrewdriverSmall screwdriver to easily DescriptionUse in combination with the KitAlysis Bench Top Inertion Box (Z742064) or a glove box/glove bag to provide inert atmosphere for kit set-up.Designed to be used with KitAlysis High-Throughput Screening Kits.Components:24-Well Reaction BlockTorque ScrewdriverSmall screwdriver to easily remove torqued screws after reaction is complete.10 Reaction Block Replacement Screws... Read More | Inquire | This reagent kit uses an adsorption column that can specifically bind to viral RNA and a unique buffer system, suitable for isolating viral RNA from cell-free body fluids such as serum, plasma, urine, cerebrospinal fluid, and cell culture supernatants. The viral RNA specifically binds to the siliconThis reagent kit uses an adsorption column that can specifically bind to viral RNA and a unique buffer system, suitable for isolating viral RNA from cell-free body fluids such as serum, plasma, urine, cerebrospinal fluid, and cell culture supernatants. The viral RNA specifically binds to the silicon substrate membrane, and pollutants flow through the membrane. Completely remove impurities such as proteins through two efficient washes, and then wash high-purity viral RNA with RNase free water or RNase Free Water provided by the reagent kit. The virus RNA extracted by this kit can be directly used for experiments such as RT-PCR, Real time RT-PCR, and Western blot analysis. R666005Component50 TStorageR666005ABuffer GL15 mLRTR666005BBuffer RW140 mLRTR666005CBuffer RW2（concentrate）11 mLRTR666005DProteinase K12.5 mgRTR666005EProteinase K Storage Buffer1.25 mLRTR666005FRNase-Free Water10 mLRTR666005GSpin Columns RS with Collection Tubes50 setsRTR666005HRNase-Free Centrifuge Tubes（1.5 mL）50 EART Self prepared reagent: anhydrous ethanol, 0.9% NaCl.Preparation and important precautions before the experiment1. Add 1.25 ml of Protein K Storage Buffer to Protein K to dissolve it and store at -20 ℃. The prepared Protein K should not be left at room temperature for a long time to avoid repeated freeze-thaw cycles, which may affect its activity.2. To prevent RNase pollution, attention should be paid to the following aspects:1) Use RNase free plastic products and gun heads to avoid cross contamination.2) Glassware should be dry baked at a high temperature of 180 ℃ for 4 hours before use, while plastic containers can be soaked in 0.5 M NaOH for 10 minutes, thoroughly rinsed with water, and then sterilized under high pressure.3) Prepare the solution using water without RNase.4) Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.3. Serum or plasma should avoid repeated freeze-thaw cycles that may cause protein denaturation or precipitation, reduce viral titers, and thus affect the yield of extracted viral nucleic acids.4. Before the first use, anhydrous ethanol should be added to Buffer RW2 according to the instructions on the reagent bottle label.5. If buffer GL precipitates, it can be heated at 56 ℃ to dissolve and then placed at room temperature.6. All centrifugation steps should be carried out at room temperature unless otherwise specified, and all operation steps should be carried out quickly.Operation steps1. Take 200 at room temperature µ Add serum or plasma to a 1.5 ml centrifuge tube (self provided). Attention: Less than 200 µ 0.9% NaCl (provided by the customer) can be added to make up for it.2. Add 20 to the solution in the previous step µ Protein K, mix well.3. Add 200 µ L Buffer GL, vortex oscillation for 15 seconds. Note: Do not directly add Protein K to Buffer GL. 4. Incubate at 56 ℃ for 15 minutes, briefly centrifuge, and collect the solution on the tube wall to the bottom of the tube.5. Add 250 µ Anhydrous ethanol, vortex for 15 seconds, incubate at room temperature for 5 minutes, briefly centrifuge, and collect the solution from the tube wall to the bottom of the tube.6. Add all the solution obtained in step 5 to the Spin Columns RS that have been loaded into the collection tube. If it is not possible to add all the solution to the adsorption column at once, please transfer it in two batches, centrifuge at 12000 rpm (~13400 × g) for 1 minute, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.7. Add 500 to the adsorption column µ Centrifuge at 12000 rpm for 1 minute, discard the waste liquid from the collection tube, and place the adsorption column back into the collection tube.8. Add 500 to the adsorption column µ Buffer RW2 (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.9. Add 500 to the adsorption column µ Centrifuge anhydrous ethanol at 12000 rpm for 1 minute, discard the waste liquid from the collection tube, and place the adsorption column back into the collection tube. 10. Centrifuge at 12000 rpm for 3 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes to thoroughly air dry.Attention:1) The purpose of this step is to remove residual ethanol from the adsorption column, which will affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).2) Recommended steps: Place the adsorption column into a new 1.5 ml centrifuge tube (provided), open the tube cover, and incubate in a 56 ℃ oven for 3 minutes to thoroughly dry the membrane of the adsorption column.11. Place the adsorption column in a new RNase free centrifuge tube and add 20-50 to the middle of the adsorption column in the air µ Place RNase Free Water at room temperature for 5 minutes, centrifuge at 12000 rpm for 1 minute, collect RNA solution, and store RNA at -70 ℃ to prevent degradation.Attention:1) The volume of RNase Free Water should not be less than 20 µ l. Small volume affects the recovery rate.2) If you want to increase RNA production, you can use 20-50 µ Repeat step 11 for the new RNase Free Water.3) If you want to increase the RNA concentration, you can add the obtained solution back to the adsorption column and repeat step 11... Read More | Product contentComponentY665957-1mlY665957-5ml2×GoldStar Probe Mixture1 ml5×1 mlProbe Primer Mix300 µl5×300 µlHuman DNA Standard（100 ng/µl）100 µl5×100 µl50×High ROX40 µl200 µlProduct IntroductionThis product is a real-time Product contentComponentY665957-1mlY665957-5ml2×GoldStar Probe Mixture1 ml5×1 mlProbe Primer Mix300 µl5×300 µlHuman DNA Standard（100 ng/µl）100 µl5×100 µl50×High ROX40 µl200 µlProduct IntroductionThis product is a real-time fluorescence quantitative PCR kit for detecting the concentration of human male Y chromosome, including carefully optimized PCR reaction solution, primer mixture and standards, especially suitable for the quantitative detection of precious and micro DNA samples. The kit adopts a new efficient and fast hot-start amplification enzyme GoldStar Taq DNA Polymerase, which effectively avoids non-specific amplification caused by non-specific binding of primers and templates or primer dimerization at room temperature. This product realizes accurate quantification of Y chromosome and can be applied in various fields such as genetic mapping, species polymorphism research, disease gene localization, paternity testing and forensic analysis.ROX dye is used to correct the fluorescence signal error generated between wells of a quantitative PCR instrument, and is generally used in Real Time PCR amplifiers from ABI, Stratagene, and other companies. The excitation optics vary from instrument to instrument, so the concentration of ROX dye must be matched to the corresponding fluorescence quantitative PCR instrument.Instruments that do not require ROX calibration: Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96, etc.Instruments requiring Low ROX calibration: ABI Prism7500/7500 Fast, QuantStudio®3 System, QuantStudio®5 System, QuantStudio®6 Flex System, QuantStudio®7 Flex System, ViiA 7 System, Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000, and others.Instruments requiring High ROX calibration: ABI Prism7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, etc.Note: High Rox and Low Rox are formulated as described in Method of Use 3.Scope of applicationThis product is suitable for quantitative testing of male Y chromosome DNA in scientific research, clinical, forensic medicine and paternity testing.Usage1. Amplification template preparationThe library samples to be detected were diluted with TE (10 mM Tris-Cl, pH 8.0, 1 mM EDTA), and the concentration after dilution was as close as possible to the range of 0.05-10 ng/µL. 4°C on ice was set aside.2. Standard dilution: according to the following table, firstly dilute Human DNA Standard (100ng/uL) with TE to make 5 standards of different concentrations according to the table below. 10ng/µL of DNA Standard 1 (Std.1) can be stored stably at -20℃ for 1 month; Std2-5 can only be used on the same day, and should be placed at 4℃ or on ice when not in use for the time being after preparation. When Std2-5 are not used temporarily after preparation, they should be stored at 4℃ or on ice.Standard sampleCorresponding concentration(ng/µl)Minimum Dilution Volume (Unit：µl）Std.11010 [100 ng/µl DNA Standard]+ 90 TEStd.22.520 [Std. 1] +60 TEStd.30.62520 [Std. 2] +60 TEStd.40.1562520 [Std. 3] +60 TEStd.50.039062520 [Std. 4] +60 TE3. qPCR reaction system preparationBefore preparation, the cryopreserved reagents to be used were completely melted and mixed by inverting several times, then centrifuged briefly and prepared. Standards and templates were diluted as described above and prepared.The base reaction system for 20 µL was as follows:Reagent20 µl Reaction system2×GoldStar Probe Mixture10 µlProbe Primer Mix3 µlTemplate4 µlddH₂O3 µlNote: High ROX model: add 1 µL of 50×High ROX per 50 µL of reaction system; Low ROX model: add 1 µL of 50×High ROX per 500 µL of reaction system.A sufficient amount of reaction system mixture was prepared according to the need, and after the reaction system was prepared and mixed thoroughly, it was added to the reaction wells in a volume of 16 µl per well. Then add the prepared standards and diluted samples into the corresponding reaction wells, the amount of addition is 4µL/well. TE was added to the blank control tube, and the same amount was added at 4 µL/well.It is recommended to use 20 µL for the reaction, if you need to perform a smaller system reaction, reduce the system components in equal proportion.4. qPCR reaction programThe PCR mix of this kit contains a FAM fluorescent probe for the target gene and a VIC fluorescent probe with internal reference to Internal PCR Control (IPC). qPCR program with dual fluorescence of hydrolyzed probes needs to be selected for the assay. Please follow the instructions of the instrument used to set up the qPCR program, and the PCR temperature conditions are as follows:1. Standard curve productionThe standard curve was plotted with reference to the Excel sheet for data processing. The correlation coefficient R2 of the standard curve should be not less than 0.98, and the slope should be located between -3.1 and -3.6 when the Ct value is used as the longitudinal coordinate. If the parameters of the standard curve are unreasonable, it is recommended to repeat the experiment.DNA Standard NameDNA Standard Concentration（ng/µL）DNA Standard 110DNA Standard 22.5DNA Standard 30.625DNA Standard 40.15625DNA Standard 50.03906252. Analysis of results and calculation of concentrationsThe Ct difference between experimental replicate wells for FAM signaling of the target gene should be no more than 0.3, otherwise invalid data need to be deleted or the experiment needs to be repeated, do not use Ct outside the valid Ct range of the standard curve to calculate the concentration of the sample.For specific calculations, please refer to the data processing Excel for this product.If the FAM signal is abnormal, the VIC signal of the internal reference Internal PCR Control (IPC) needs to be analyzed to confirm whether the PCR reaction process is abnormal. If the Ct value of the sample null VIC is significantly larger than that of the standard or blank control wells, it means that the sample inhibits the PCR reaction.matters needing attention1. Before testing, these instructions should be read in detail. It should be operated by personnel with professional experience or qualified by training.2. For use, please mix gently by turning up and down, avoid foaming as much as possible, and use it after centrifugation for a short period of time.3. Avoid repeated freezing and thawing of the product, repeated freezing and thawing may degrade the performance of the product.4. When preparing the reaction solution, please use new or non-contaminated tips and centrifuge tubes to prevent contamination as much as possible... Read More |