| Description | Product Introduction:This product is a one-stop processing kit from plasma to peptides. It utilizes nanotechnology to effectively enrich low-abundance proteins in plasma and processes plasma proteins in combination with the innovative concept of "All In One Tube". Even researchers without omics Product Introduction:This product is a one-stop processing kit from plasma to peptides. It utilizes nanotechnology to effectively enrich low-abundance proteins in plasma and processes plasma proteins in combination with the innovative concept of "All In One Tube". Even researchers without omics background can quickly prepare pretreated samples, which can be directly used for subsequent mass spectrometry analysis.Product Components and Storage Conditions:P1456360Component10T25TStorageP1456360AMagnetic beads10T25T4℃P1456360BIncubation Buffer I4.8 mL12 mLRTP1456360CIncubation Buffer II6 mL15 mLRTP1456360DWashing Buffer6 mL15 mLRTP1456360ELysis Buffer0.6 mL1.5 mL-20℃. Store in the dark.P1456360FBalance Buffer2.8mL7 mL4℃P1456360GDigest Buffer16 µL40 µL-20℃P1456360HStop Buffer300 µL750 µLRTP1456360IWash Buffer I4 mL10 mLRTP1456360JWash Buffer II2.4 mL6 mLRTP1456360KWash Buffer III2.4 mL6 mLRTP1456360LElution Buffer4.8 mL12 mLRT. Store in the dark.P1456360MLoading Buffer120 µL300 µLRTP1456360NTip柱10T25TRTProduct Features:Convenient and fast: The processing from plasma to peptides can be completed with one kit.Simple operation: The instruments used are simple, requiring only a conventional centrifuge and a metal bath.Good stability: Each batch undergoes strict quality inspection, ensuring high reproducibility of experimental results.Operating Procedure: 1.Centrifuge the plasma sample (3000g, 10min), and take the supernatant for later use (if storage is required, store it at -80°C for long-term preservation to avoid repeated freezing and thawing).2.Take 50-100µL of centrifuged plasma, add 400µL of Incubation buffer I, then add 30µL of Magnetic beads, vortex to mix, and incubate at room temperature on a shaking mixer for 1 hour.3.After incubation, use a magnetic rack to magnetically separate for 3min, and discard the supernatant.4.Remove the EP tube, add 500µL of Incubation buffer II, gently invert up and down to mix several times, magnetically separate for 3min, and discard the supernatant.5.Remove the EP tube, add 500µL of Washing Buffer, gently invert up and down to mix several times, magnetically separate for 3min, and discard the supernatant.6.Remove the EP tube, add 50µL of Lysis Buffer to resuspend the beads, place in a water bath at 95°C for 10min, then take it out and cool to room temperature.7.Add 225µL of Balance buffer to the EP tube that has cooled to room temperature.8.Add 1µL of Digest buffer to the sample, and perform enzymatic digestion with shaking in a metal bath at 37°C and 1200rpm for 3-16h.9.After enzymatic digestion, take out the EP tube, add 25µL of Stop buffer to the sample, and vortex to mix.10.Add 320µL of Wash buffer I, shake vigorously for 3min, centrifuge at 15000rpm for 3min, and remove the upper liquid.11.Transfer the lower layer sample into the Tip column, centrifuge at 2500rpm for 3-5min until all the liquid is centrifuged down. If the liquid flow rate is slow, the rotation speed can be appropriately increased.12.Add 200µL of Wash buffer II (shake for 10-20s before use) to the desalting column, centrifuge at 2500rpm for 3-5min until all the liquid is centrifuged down.13.Add 200µL of Wash buffer III to the desalting column, centrifuge at 2500rpm for 3-5min until all the liquid is centrifuged down.14.Put the desalting column into a new EP tube, add 200µL of Elution buffer to the desalting column, centrifuge at 2000rpm for 3-5min until all the liquid is centrifuged down.15.Repeat the previous step, collect the eluates from both times, and freeze-dry them.16.Add 10µL of Loading buffer, vortex vigorously for 3min, centrifuge at 2000g for 10min, take an appropriate amount of sample for mass spectrometry detection. Taking the HF-X instrument as an example, 0.5-1µg of sample is sufficient for loading. Automated Operation Process:It is compatible with mass spectrometry proteomics pretreatment workstations, enabling one-stop processing from plasma to peptides. This eliminates manual operation errors, improves the reproducibility of sample preparation workflows, and provides high precision and reliability for various laboratory procedures... Read More | Inquire | Inquire | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | 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 |