| Description | This product is a reagent kit for reverse transcription of genomic DNA, which can remove genomic DNA in 2 minutes at 42 ℃. Meanwhile, the reverse transcription reagent contains components that inhibit gDNA Remover, and the samples treated with gDNA Remover can be directly subjected to reverse This product is a reagent kit for reverse transcription of genomic DNA, which can remove genomic DNA in 2 minutes at 42 ℃. Meanwhile, the reverse transcription reagent contains components that inhibit gDNA Remover, and the samples treated with gDNA Remover can be directly subjected to reverse transcription reaction to synthesize cDNA.This reagent kit is equipped with a novel and efficient reverse transcriptase HiFiScript, which significantly enhances the transcriptional activity of the enzyme through novel mutation sites. Meanwhile, reverse transcription reaction only takes 15 minutes to complete the synthesis of the first strand of cDNA. 5 × HifiScript RT MasterMix is a reverse transcription premix that contains all the reagents required for reverse transcription, making it easy and fast to operate.H665909Component100 TStorageH665909A10×gDNA Remover Mix 100 µL-20℃. Avoid freeze/thaw cycle.H665909B5×HiFiScript RT MasterMix 400 µL-20℃. Avoid freeze/thaw cycle.H665909CRNase-Free Water 1.5 mL-20℃. Avoid freeze/thaw cycle. Product features1. Quick genome removal: gDNA Remover containing genomic DNA can remove genomic DNA in just 2 minutes.2. Rapid reverse transcription: cDNA first strand synthesis can be completed in 15 minutes.3. Convenient and fast: Ready to use reverse transcription Mix, easy to operate.4. High sensitivity: cDNA first strand can be synthesized using pg level total RNA or mRNA templates.5. Efficient reverse transcription efficiency: Novel mutation sites significantly enhance enzyme activity performance, resulting in higher yields of cDNA.Matters needing attention1. During the operation, RNase contamination should be avoided to prevent RNA degradation or cross contamination during experiments. It is recommended that operators wear masks and disposable gloves, frequently change gloves, and use specialized instruments and consumables.2. Disposable plastic containers should be used as much as possible for experiments. If glassware is used, a 0.1% DEPC (diethyl carbonate 1. ester) aqueous solution should be treated at 37 ℃ for 12 hours, and then sterilized under high pressure at 120 ℃ for 30 minutes before use. Alternatively, glassware should be sterilized under dry heat at 180 ℃ for 60 minutes before use. The sterile water used in the experiment should be treated with 0.1% DEPC and then subjected to high-pressure sterilization.3. The reverse transcription system is prepared on ice for operation to prevent RNA degradation. The enzyme in the reagent kit should be stored at -20 º C as soon as possible after use, and repeated freeze-thaw should be avoided as much as possible.UsageThaw the template RNA on ice; After thawing at room temperature, immediately place the components of the reagent kit on ice. Before use, mix each solution by vortex oscillation and centrifuge briefly before use.1、 Genomic DNA removal reaction1. Prepare a reaction system on ice according to the following table, with a total volume of 10 µ L. To ensure the accuracy of the preparation of the reaction solution, a pre mixed system is first prepared in the amount of reaction number+2, then divided into each reaction tube, and finally RNA samples are added. Reagent 10 µl Reaction system 10×gDNA Remover Mix 1µl RNA Template¹ 10 pg-1 µg RNase-Free Water up to 10 µl Note: 1) If the total RNA amount is greater than 1 µ g, please expand the reaction system proportionally.2. Vortex shake and mix well, briefly centrifuge to collect the solution on the pipe wall to the bottom of the pipe. 3. Incubate at 42 ℃ for 2 minutes (during room temperature reaction, it can be extended to 30 minutes).4. After the reaction is complete, centrifuge briefly and cool on ice.2、 Reverse transcription reaction1. Prepare the reaction system on ice according to the following table, and prepare the reaction solution on ice. To ensure the accuracy of the reaction solution configuration, first prepare a premixed solution in the amount of reaction number+2, and then divide it into 10 batches µ Take 10% of the prepared premix from each reaction tube µ Add to the reaction tube of step 1 that has completed genome removal. Reagent 20 µl Reaction system Step 1 Reaction solution 10µl 5×HiFiScript RTMaster Mix 40µl RNase-Free Water 60µl 2. Mix well and centrifuge briefly to collect the solution on the tube wall to the bottom of the tube.3. cDNA synthesis reaction conditions: Incubate at 37 ℃ for 15 minutes and 85 ℃ for 5 seconds.4. After the reaction is completed, centrifuge briefly and place it on ice before proceeding with the subsequent reaction. If it needs to be stored for a long time, please place it at -20 ℃... Read More | Inquire | 1、Product attributeShelf life: 24 monthsReaction time：long (up to 45 minutes) at 20-37°CLot-to-lot variation：<10%Boiling point : 100℃pH-Value (at 20 °C): 9.0-9.8 Density (20℃) : 1.0302 g/cm³Water solubility: easily solubleAppearance: colourless to 1、Product attributeShelf life: 24 monthsReaction time：long (up to 45 minutes) at 20-37°CLot-to-lot variation：<10%Boiling point : 100℃pH-Value (at 20 °C): 9.0-9.8 Density (20℃) : 1.0302 g/cm³Water solubility: easily solubleAppearance: colourless to light yellow liquidOdour: odourlessIncubation temperature: 20-37 °CLight sensitiveHeat sensitive 2、Requirements for storage rooms and vessels1.Keep container tightly closed.2.Keep cool. protected from light3. Do not store together with: Oxidizing agent. 4. Contaminated or leaked out substrate solution from damaged bottles should not be used anymore and has to be destroyed.5. Use isolated containers with some cool bags for transport.6. Spontaneous decay will increase the background. If stored at room temperature, the velocity of the decay will increase. Thus, both storage and transport at room temperature should be avoided. Nevertheless, the activity of the solution is not affected by storage at room temperature. The solution still works beyond the expiry date, but some applications, especially those including visual evaluation, may be hampered by increased background. 3、Effective Components and Principle of FunctionIn different buffer solutions (pH = 9.5), with supplementation if required, the effective componentpara nitrophenyl phosphate (pNPP) is dissolved. Alkaline Phosphatase transfers the phosphate residue to an acceptor. Under alkaline conditions a yellow colour occurs, resulting from the formed nitrophenol. 4、Biosafety informationThis mixture is not classified as hazardous in accordance with Regulation (EC) No 1272/2008； 5、Advantage1. Signal yield comparable to competitor ready-to-use solutions2. Broad measurement range3. Very low background signals4. Very low blank drift during long-term storage (<0.15 AU within 24 months)5. High colour stability after reaction stop with this product and other commonly used stopping solutions 6、Instruction for usageFor bottling consider the following instructions:• Work in a dust free and darkened room.• Keep the solution as cool as possible.• Avoid contact of the solutions with any metal parts• Clean all instruments and vessels very extensively.• Wear powder-free gloves during bottling.• Close the bottles immediately to minimize the influence of light and dust.• Use clean bottles that are impermeable to light made from HDPE or PP. 7、 General Instructions for the Use in Blotting Systems Only qualified laboratory staff, who are familiar with the basics of immunological methods, are allowed to use these solutions.The substrate solutions can be used in qualitative and quantitative ELISA procedures.When using 96-well microtiter plates, adding 100 µL of substrate per well after incubation and washing is recommended. After substrate incubation the reaction can be stopped and the photometric measurement can be carried out. Using higher incubation temperatures (37° C) may shorten the incubation time. The reaction can be stopped by using the special developed solution stop. The use of other commercially available stop solutions cannot safely exclude a further increase of the signal. Addition of a stopping solution does not change the general shape of the spectrum. The unstopped and the stopped solution should be measured at 405 nm and the background correction: should be measured at 620 nm... Read More | ProductsThis product is a high purity genomic DNA extract from 293T cells, agarose gel (0.7%) electrophoresis showed that the size of the DNA extract is more than 15Kb, and basically no degradation, the product is ultimately preserved in TE Buffer, which can be widely used in molecular biology ProductsThis product is a high purity genomic DNA extract from 293T cells, agarose gel (0.7%) electrophoresis showed that the size of the DNA extract is more than 15Kb, and basically no degradation, the product is ultimately preserved in TE Buffer, which can be widely used in molecular biology experiments, such as PCR, enzyme digestion, hybridization, microarray analysis, and other molecular biology experiments.The product was quantified using NanoDrop One at a concentration of 200 ng/µL.Preparation and precautions before useLong-term storage at -20˚C is recommended. Before use, the bottle should be removed from the refrigerator and equilibrated to room temperature and centrifuged before opening the cap for use. Samples should be restored to the sealed state as soon as possible after opening.How to use (take qPCR experiment as an example)1. Amplification template preparationThe 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. The samples were placed on ice at 4°C and set aside.2. Standard dilution: according to the following table, firstly dilute Human DNA Standard 1 (100ng/uL) with TE to make 5 different concentrations of standards 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 not used temporarily after preparation, it should be stored at 4℃ or on ice.styleCorresponding concentration (ng/µL)Minimum dilution volume (in µL)Std.11010 [100 ng/µL DNA Standard 1] + 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 preparationThe cryopreserved reagents to be used were completely thawed and mixed by inversion several times before preparation, and then briefly centrifuged and prepared for use. 20 µL of the base reaction system was as follows.The base reaction system for 20 µL was as follows:reagents20µL reaction system2×qPCRMix10µLPrimerMixXµLProbeMixXµLTemplate4µLddH2OMake up to 20 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.Usually, better results can be obtained with a primer concentration of 0.2 µM, and 0.1-1.0 µM can be used as a reference for setting the range.The concentration of the probe used is related to the fluorescent quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance, so please refer to the manual of the instrument or the specific requirements for the use of each fluorescent probe for the adjustment of the concentration during actual use.Prepare a sufficient amount of reaction system mixture as required. After the reaction system has been prepared and mixed thoroughly, add 16 µL per well to the reaction wells. Then add the prepared standard and diluted sample into the corresponding reaction wells, the volume of addition is 4µL/well. TE was added to the blank control tube, and the same amount of TE 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 following is an example of our GoldStar Probe Mixture reaction conditions, which should be improved and optimized according to the PCR product template, primer structure and target fragment size.movetemptimingcirculatepremutability95°C10min1denaturation95°C10sec55Annealing/Extension60°C30sec5Data analysis1. 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 not be lower than 0.98, and the slope should be between -3.1 and -3.6 when the Ct value is the vertical coordinate. If the parameters of the standard curve are unreasonable, it is recommended to repeat the experiment... Read More | Inquire |