| Description | 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 | Products B669892Component50 TStorageB669892ABuffer RCL3×260 mL2-8℃B669892BBuffer GR25 mLRTB669892CBuffer GL25 mLRTB669892DBuffer GW1 (concentrate)13 mLRTB669892EBuffer GW2 (concentrate)15 mLRTB669892FBuffer GE15 mLRTB669892GProteinase K50 mgRTB669892HProteinase K Storage Buffer5 Products B669892Component50 TStorageB669892ABuffer RCL3×260 mL2-8℃B669892BBuffer GR25 mLRTB669892CBuffer GL25 mLRTB669892DBuffer GW1 (concentrate)13 mLRTB669892EBuffer GW2 (concentrate)15 mLRTB669892FBuffer GE15 mLRTB669892GProteinase K50 mgRTB669892HProteinase K Storage Buffer5 mLRTB669892ISpin Columns DL with Collection Tubes50 setsRTProductsThis kit is suitable for the extraction of total DNA, including genomic DNA, mitochondrial DNA and viral DNA, from fresh or frozen whole blood (blood samplestreated with anticoagulants such as citrate, EDTA or heparin), plasma, serum, haematocrit brown and yellow layers, bone marrow, cell-free body fluids, etc. Theproduct can process 1-5 ml of whole blood, and can be purified to obtain sizes rangingfrom 100bp to 50kb. The purified DNA is of high yield and good quality, with maximumremoval of proteins, pigments, lipids and other inhibitory impurities, and can bedirectly used in PCR, fluorescence quantitative PCR, enzyme digestion and SouthernBlot.Self-contained reagent: anhydrous ethanol.Pre-experiment Preparation and Important Notes1. Add 5ml Proteinase K Storage Buffer to Proteinase K to dissolve it, and storeit at -20℃. Do not leave the prepared Proteinase K at room temperature for a longtime, and avoid repeated freezing and thawing to avoid affecting its activity.2. Repeated freezing and thawing of the sample should be avoided, as this may resultin smaller DNA fragments and a decrease in the amount of extracted DNA. 3.This kit can extract up to 1-5 ml of whole blood samples, if you need to extracta large number of blood samples, please use the blood genome non-column extractionkit. 4. Anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to theinstructions on the label of the reagent bottle before first use.5. Please check Buffer GL for crystallization or precipitation before use, if thereis any crystallization or precipitation, please put it in 56℃water bath to re-dissolve.6. If the downstream experiments are sensitive to RNA contamination, 4µl of DNaseFree RNase A (100mg/ml) can be added, RNase A is not provided in the kit, and canbe ordered separately from our company if needed.7. The Buffer RCL in the kit cannot be used further after turbidity.procedure1. Add 1-5 ml of blood sample to a centrifuge tube (supplied) and add 3 times thevolume of Buffer RCL and gently vortex or invert to mix.2. Centrifuge at 3000 rpm (~900 x g) for 10 minutes and carefully aspirate thesupernatant.3. Add 400 µl Buffer GR to the precipitate and resuspend the precipitate. Note: If the downstream assay is sensitive to RNA, add 4 µl of RNase A (100 mg/ml)solution, shake for 15 seconds, and leave at room temperature for 5 minutes.4. For 1-2 ml blood sample extraction, add 40µl Proteinase K to the above solutionand mix well; for 2-5 ml blood sample extraction, add 100µl Proteinase K to theabove solution and mix well.5. Add 400 µl of Buffer GL, mix upside down 15 times, and vigorously vortex andshake for at least 1 minute. Note: Do not add Proteinase K directly to Buffer GL.6. Incubate at 70°C for 10 minutes, during which time mixing was inverted severaltimes.Note: 1) If the solution is not completely clear, add appropriate amount of Proteinase K and incubate. Extend the incubation time until the solution is completely clear. 2) The yield of DNA has been maximized by 10 minutes of incubation, and continuedprolongation of the incubation time has no effect on DNA yield or purity.7. Add 400 µl of anhydrous ethanol and mix upside down 10 times. Centrifuge brieflyto concentrate the liquid on the walls and cap to the bottom of the tube.8. Add all of the solution obtained in the previous step to the Spin Columns DL inthe collection tube. If the solution cannot be added all at once, transfer it severaltimes. centrifuge at 12,000 rpm (~13,400 x g) for 1 minute, pour off the waste liquidfrom the collection tube, and put the column back into the collection tube.9. Add 500 µl of Buffer GW1 to the adsorption column (check that anhydrous ethanolis added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquidin the collection tube, and put the adsorption column back into the collection tube.Note: It is recommended that step 9 be repeated if the sample being extracted isthe blood genome of a species such as mice or monkeys from which hemoglobin isdifficult to remove.10. Add 500 µl Buffer GW2 to the adsorption column (check that anhydrous ethanolis added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquidin the collection tube, and put the adsorption column back into the collection tube.Note: Step 10 can be repeated if further DNA purity is required.11. Centrifuge at 12,000 rpm for 2 minutes and pour off the waste liquid in thecollection tube. Leave the adsorption column at room temperature for several minutesto dry thoroughly. Note: The purpose of this step is to remove residual ethanol from the adsorptioncolumn, which can interfere with subsequent enzymatic reactions (digestion, PCR,etc.)12. Place the adsorption column in a new centrifuge tube, add 50-200 µl of BufferGE or sterilized water to the middle of the adsorption column overhanging the column,leave it at room temperature for 2-5 minutes, centrifuge at 12,000 rpm for 1 minute,collect the DNA solution, and store the DNA at -20℃.Note: 1) If the downstream experiment is sensitive to pH or EDTA, you can use sterilized water for elution. The pH of the eluent has a great influence on theelution efficiency, if water is used as the eluent should ensure that its pH is7.0-8.5 (you can use NaOH to adjust the pH of the water to this range), and the elutionefficiency is not high when the pH is lower than 7.0.2) Incubation at room temperature for 5 minutes prior to centrifugation increasesyield.3) Re-elution with an additional 50-200 µl Buffer GE or sterilized water can increase the yield.4) If the final concentration of DNA is to be increased, the DNA eluate obtainedin step 12 can be re-spiked onto the adsorbent membrane and centrifuged at 12,000rpm. 1min; if the elution volume is less than 200µl, the final concentration of DNA canbe increased, but the total yield may be reduced. If the amount of DNA is less than1 µg, elution with 50 µl Buffer GE or sterilized water is recommended.5) Because DNA preserved in water is subject to acidic hydrolysis, for long-termstorage, it is recommended that it be eluted with Buffer GE and stored at -20℃... Read More | DescriptionProvides an inert environment to run oxygen sensitive cross-coupling reactions in a laboratory fume hood.Designed to be used with KitAlysis High-Throughput Screening Kits and KitAlysis 24-Well Reaction Block | O665690 Component 50T Storage O665690A DNase I 1000 U -20℃.Avoid freeze/thaw cycle. O665690B 10×Reaction Buffer 1000 µL -20℃.Avoid freeze/thaw cycle. O665690C Buffer RLS 40 mL RT O665690D Buffer RW1 40 mL RT O665690E Buffer RW2 (concentrate) 11 mL RT O665690F RNase-Free Water O665690 Component 50T Storage O665690A DNase I 1000 U -20℃.Avoid freeze/thaw cycle. O665690B 10×Reaction Buffer 1000 µL -20℃.Avoid freeze/thaw cycle. O665690C Buffer RLS 40 mL RT O665690D Buffer RW1 40 mL RT O665690E Buffer RW2 (concentrate) 11 mL RT O665690F RNase-Free Water 10 mL RT O665690G Spin Columns FS with Collection Tubes 50 EA RT O665690H Spin Columns RM with Collection Tubes 50 EA RT O665690I RNase-Free Centrifuge Tubes (1.5 mL) 50 EA RTProduct IntroductionThis kit is suitable for extracting RNA from a wide range of plants, even from plants rich in polysaccharides and polyphenols, high quality RNA can be successfully extracted, such as rice leaves, wheat leaves, corn leaves, tobacco leaves, pine needles, ginkgo leaves, poplar leaves, pomegranate leaves, holly leaves, apples, peaches, pears, tomatoes, cherries, apricots, bananas, grapes, loquats, cinnamon rinds, cinnamon pulp, lychee fruit rinds, lychee pulp, soybean, peanut, corn, potato tuber, moonflower petal, pomegranate petal, shiitake mushroom, flat mushroom and other samples. The unique lysate formula can rapidly inactivate the RNA enzyme in the cell, effectively remove the effect of polysaccharide and polyphenol on RNA extraction, without the need for phenol, chloroform and other reagents, while using silicon matrix membrane adsorption of RNA for purification, the total RNA extracted is highly pure, without the contamination of genomes, proteins and other impurities, and can be used for Real Time RT-PCR, RT-PCR, It can be used for Real Time RT-PCR, RT-PCR, Northern Blot, Dot Blot, in vitro translation and other downstream experiments.RNA yieldSelf-contained reagents: β-mercaptoethanol, anhydrous ethanol (freshly opened or for RNA extraction)Pre-experiment Preparation and Important Notes1. To prevent RNase contamination, attention should be paid to the following aspects:1) Use RNase-free plastics and tips.(2) Operators wear disposable masks and gloves, and change gloves diligently during the experiment.2. Avoid repeated freezing and thawing of the extracted samples, otherwise it will affect the rate and quality of RNA extraction.3. If Buffer RLS produces a precipitate, heat to dissolve it and leave at room temperature.4. Please add β-mercaptoethanol to Buffer RLS before use, add 20µl β-mercaptoethanol to 1ml Buffer RLS. Buffer RLS with β-mercaptoethanol can be stored for 1 month at room temperature.5. Anhydrous ethanol should be added according to the instructions on the reagent bottle label before using Buffer RW2 for the first time. Operation steps1. Homogenization: Take 50-100mg of plant tissue and quickly grind it into powder in liquid nitrogen, add 500µl of Buffer RLS (please check whether β-mercaptoethanol is added before use), and immediately mix it by vortexing with vigorous shaking.Note: For materials that are extremely rich in water content, such as watermelon pulp, tomato, pear pulp, etc., more material can be added appropriately, up to 200 mg; for starch-rich samples or mature leaves, the amount of Buffer RLS can be increased appropriately, up to 700 µl.2. Centrifuge at 12,000 rpm (~13,400 x g) for 2 min at 4°C.3. Transfer the supernatant into the filter columns (Spin Columns FS) that have been loaded into the collection tubes, centrifuge at 12,000 rpm at 4°C for 1 minute, carefully aspirate the supernatant in the collection tubes and transfer it to new RNase-Free centrifugation tubes (self-provided), avoiding the tip of the gun from touching the cell debris precipitation in the collection tubes as much as possible.4. Slowly add 0.5 times the volume of the supernatant in anhydrous ethanol, mix well (a precipitate may appear), and transfer the resulting solution together with the precipitate to a Spin Columns RM in a collection tube, or in two batches if you cannot add all of the solution at once. centrifuge the column for 1 minute at 12,000 rpm at 4°C. Dispose of the spent solution and place the column back into the collection tube. Centrifuge at 12,000 rpm for 1 minute at 4°C, discard the spent solution and return the column to the collection tube.5. Add 350 µl of Buffer RW1 to the adsorbent column RM, centrifuge at 12,000 rpm at 4°C for 1 min, discard the waste solution and put the adsorbent column back into the collection tube.6. Preparation of DNase I mixture: Take 52µl of RNase-Free Water, add 8µl of 10×Reaction Buffer and 20µl of DNase I (1U/µl) to it, mix well, and prepare a final volume of 80µl of reaction solution.7. Add 80µl of DNase I mixture directly to the adsorption column and incubate at 20-30°C for 15 minutes.8. Add 350 µl of Buffer RW1 to the adsorbent column RM, centrifuge at 12,000 rpm at 4°C for 1 min, discard the waste solution and put the adsorbent column back into the collection tube.9. Add 500 µl of Buffer RW2 to the adsorbent column RM (check that anhydrous ethanol is added before use), centrifuge at 12,000 rpm for 1 minute at 4°C, discard the waste solution and put the adsorbent column back into the collection tube.10. Repeat step 9.11. Centrifuge at 12,000 rpm for 2 minutes at 4°C.Note: The purpose of this step is to remove residual ethanol from the adsorption column; ethanol residue can interfere with subsequent enzymatic reactions (zymography, PCR, etc.).12. Load the adsorption column RM into new RNase-Free Centrifuge Tubes (1.5 ml), add 30-50 µl of RNase-Free Water dropwise to the middle part of the adsorption membrane overhang, leave it at room temperature for 2 min, and centrifuge at 12,000 rpm at 4°C for 1 min, and store the resulting RNA solution at -70°C to prevent degradation.Note: 1) The volume of RNase-Free Water should not be less than 30 µl, too small volume affects the recovery rate.2) If you want to increase the RNA yield, repeat step 12 with 30-50 µl of fresh RNase-Free Water.3) If the RNA concentration is to be increased, the resulting solution can be reintroduced into the adsorption column and step 12 repeated... 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 |