| Description | Inquire | D665729 Component 50 T Storage D665729A Conversion Buffer CR 5×1 mL RT D665729B Buffer CL 30 mL RT D665729C Buffer MD 0.4 mL RT D665729D Buffer DB 10 mL RT D665729E Buffer WB (concentrate) 10 mL RT D665729F Buffer GW1 (concentrate) 13 mL RT D665729G Buffer GW2 (concentrate) 15 mL RT D665729H D665729 Component 50 T Storage D665729A Conversion Buffer CR 5×1 mL RT D665729B Buffer CL 30 mL RT D665729C Buffer MD 0.4 mL RT D665729D Buffer DB 10 mL RT D665729E Buffer WB (concentrate) 10 mL RT D665729F Buffer GW1 (concentrate) 13 mL RT D665729G Buffer GW2 (concentrate) 15 mL RT D665729H Buffer EB 4 mL RT D665729I Buffer PS 10 mL RT D665729J Spin Columns DF 50 Pcs 2-8 ℃ D665729K Collection Tubes 50 Pcs RTProduct Introduction:The basic principle of this reagent kit is that after DNA is treated with sodium bisulfite, unmethylated cytosine can be transformed into uracil, while methylated cytosine remains unchanged. And adopting an innovative high-temperature treatment method, the transformation time is greatly shortened, the transformation efficiency is improved, and the transformation efficiency can reach over 99%. At the same time, using a silicon-based membrane purification column, DNA can be recovered and purified from the methylated solution through a simple binding washing elution step. The recovered DNA has high purity and good integrity, and can be directly used for sequencing, methylated PCR detection, chip analysis, connection and transformation, enzyme digestion, labeling, microinjection, PCR and in vitro transcription and other molecular biology experiments.Self prepared reagents: anhydrous ethanol, 75% ethanol.Preparation and important precautions before the experiment1. Product usage method:(1) 10 times packaging preparation method: CT Conversion Agent is a solid mixture that must be prepared before first use. Add 2 ml sterile water and 100 µ M-Dissolving Buffer and 300 µ Add M-Diffusion Buffer to the CT Conversion Agent tube. Dissolve at 55 ° C and shake until completely dissolved. Store the CT Conversion Agent solution at room temperature (20 ° C-30 ° C) in the dark before use. The CT Conversion Agent for each tube is designed for 10 DNA treatments. In order to achieve better results, the prepared CT Conversion Agent should be used immediately. If not used immediately, the CT Conversion Agent solution can be stored at -20 ° C for 1 week. Before use, be sure to thaw the stored CT Conversion Agent solution at room temperature and mix thoroughly by shaking or inverting for 2 minutes, CT Conversion Reagent is sensitive to light, so it is important to minimize exposure to light as much as possible.(2) 50 times packaging preparation method: CT Conversion Agent and M-Dissolving Buffer are solid mixtures that must be prepared before first use. Add 5 ml of sterile water to the M-Dissolving Buffer and shake to dissolve. After all the solids have dissolved, transfer all the solution from the M-Dissolving Buffer tube to the CT Conversion Agent tube and add 5.5 ml of sterile water. Add 1.5 ml of M-Dilution Buffer to the CT Conversion Agent tube. Dissolve at 55 ° C and shake until completely dissolved. Store the CT Conversion Agent solution at room temperature (20 ° C-30 ° C) in the dark before use. The CT Conversion Agent for each tube is designed for 50 DNA treatments. In order to achieve better results, the CT Conversion Agent should be used immediately after preparation. If not immediately used, the CT Conversion Agent solution can be stored at -20 ° C for 1 week. Before use, be sure to thaw the stored CT Conversion Agent solution at room temperature and mix thoroughly by shaking or inverting for 2 minutes, CT Conversion Reagent is sensitive to light, so it is important to minimize exposure to light as much as possible.2. Before the first use, anhydrous ethanol should be added to the M-Wash Buffer according to the instructions on the reagent bottle label.Operation stepsThe range of DNA prepared each time is 1 ng-4 µ Between g, the optimal amount is 500 ng-2 µ G.1. Take 20 µ Add DNA sample into centrifuge tube (self provided), and if the sample amount is insufficient, replenish with water up to 20 µ L.2. Add 2.2 to the DNA sample µ Mix the sample well with the M-Dilution Buffer of l.3.42 ℃ water bath for 30 minutes.4. Add 220 to the sample obtained from the previous step µ Prepare the CT Conversion Agent solution, mix well, and incubate in an 80 ℃ constant temperature water bath in a dark place for 60 minutes.5. Add 480 to the solution in the previous step µ M - Buffer PA, gently mix upside down.6. Column balance: Add 200 to the spin columns DS that have been loaded into the collection tube µ Centrifuge at 12000 rpm (~13400 × g) for 2 minutes, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.7.Add all the solution obtained from step 5 to the adsorption column (already loaded into the collection tube), let it stand at room temperature for 2 minutes, 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.Attention: The maximum capacity of the adsorption column is 750 µ l. If the sample volume is greater than 750 µ L can be added in batches.8. Add 500 to the adsorption column µ Centrifuge at 12000 rpm for 1 minute using M-Buffer PA, discard the waste liquid from the collection tube, and place the adsorption column in the recovery tube.9. Add 650 to the adsorption column µ M-Wash Buffer (please check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column in the collection tube.10.12000 rpm for 2 minutes, discard the waste liquid, and place the adsorption column at room temperature for a few minutes to thoroughly air dry.Note: 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.).11. Place the adsorption column into a new centrifuge tube (provided by oneself), and add 20 drops to the middle position of the adsorption membrane in the air µ M-Elution Buffer (pH 8.5), leave at room temperature for 2 minutes. Collect DNA solution by centrifugation at 12000 rpm for 1 minute.12. Collect 20 µ Add 2.2 to DNA µ M-Diffusion Buffer, let it stand at room temperature for 30 minutes.13. Add 500 to the solution µ After pre cooling anhydrous ethanol, invert and mix well, and place the solution at -20 ℃ to precipitate for 30 minutes (overnight precipitation is more effective).14.12000 rpm for 15 minutes and gently discard the supernatant.15. Add 75% ethanol, centrifuge at 12000 rpm for 1 minute, pour out the supernatant, wait for ethanol to evaporate at room temperature, then add 20 µ Dissolve the M-Elution buffer and store the DNA at -20 ℃. The DNA collected in this step can be used for subsequent related experiments... Read More | Product contentG665801Component100 TStorageG665801A2×GoldStar Probe One Step Buffer1.4 mL-20℃. Avoid freeze/ Thaw cycle. Protect from light.G665801BGoldStar Probe One Step EnzymeMix100 µL-20℃. Avoid freeze/ Thaw cycle. Protect from light.G665801C50×High ROX50 µL-20&#Product contentG665801Component100 TStorageG665801A2×GoldStar Probe One Step Buffer1.4 mL-20℃. Avoid freeze/ Thaw cycle. Protect from light.G665801BGoldStar Probe One Step EnzymeMix100 µL-20℃. Avoid freeze/ Thaw cycle. Protect from light.G665801C50×High ROX50 µL-20℃. Avoid freeze/ Thaw cycle. Protect from light.G665801DRNase-Free Water1.5 mL-20℃. Avoid freeze/ Thaw cycle. Product Introduction This product is a specialized kit for one-step Real-Time RT-qPCR using the probe method (TaqMan, Molecular Beacon, etc.). When using this product for Real Time RT-qPCR reaction, reverse transcription and quantitative PCR are carried out in the same reaction system, and there is no need to add reagents or open the cap of the tube during the reaction process, which avoids contamination and improves the experimental efficiency at the same time. With high detection sensitivity, strong fluorescence signal and high signal-to-noise ratio, this product is very suitable for the detection of RNA viruses and other trace RNA. The special buffer system contained in this product can maximize the effectiveness of reverse transcriptase and DNA polymerase at the same time and improve the efficiency of the reaction. A wider linear range can be obtained with this product, more accurate quantification of the target gene, good reproducibility and high confidence.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 (G665836) Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96 and others. Instruments that require High ROX calibration (G665801) ABI Prism 7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, and others.matters needing attention1.Before using the reagents in this kit, please mix them gently by turning them up and down to avoid foaming as much as possible, and use them after brief centrifugation.2.This product uses RNA as the template for one-step RT-PCR experiment, RNase contamination should be avoided during operation, it is recommended to operate RNA in a special area, use special instruments and consumables, the operator with a mask and disposable gloves and often change the gloves, the experiment-related consumables should be processed with 0.1% DEPC (diethyl ether pyrocarbonate) aqueous solution for 12 hours at 37℃, and autoclaved for 30 minutes before use. The consumables should be treated with 0.1% DEPC (diethylpyrocarbonate) aqueous solution at 37℃ for 12 hours and autoclaved for 30 minutes.3.Repeated freezing and thawing of each reagent in this kit should be avoided as much as possible; repeated freezing and thawing may degrade the product performance.4.This kit must use specific primers, the choice of primers can be selected according to specific experiments, the good or bad primer design directly affects the results of RT-qPCR reaction, the design of primers need to consider the GC content, primer length, primer position, the secondary structure of the PCR product and other factors, it is recommended to use a professional primer design software for design.5.This kit is recommended to use specific probes, and it is recommended to use professional design software for designing.UsageThe following examples are conventional reaction systems and conditions, which should be improved and optimized according to the different templates, primer structures and target fragment sizes in actual operation. (Please prepare the reaction solution on ice.)1. Dissolve RNA template, primers, 2× GoldStar Probe One Step Buffer, GoldStar Probe One Step EnzymeMix and RNase-Free Water and set aside on ice.2. PCR reaction system:reagents25µl reaction systemfinal concentration2×GoldStar Probe One Step Buffer12.5µl1×Forward Primer, 10µM0.5µl0.2µM¹⁾Reverse Primer, 10µM0.5µl0.2µM¹⁾Probe, 10µM0.5µl0.2µM²⁾GoldStar Probe One Step EnzymeMix1.0µl RNA TemplateXµl10pg-100ng³⁾50 x Low ROX or High ROX (optional)⁴⁾0.5µl1×RNase-Free WaterUp to 25µlNote: 1) 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.(2) The concentration of the probe used is related to the fluorescence quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance, please refer to the instrument manual or the specific requirements for the use of each fluorescent probe for the adjustment of the concentration in actual use.(3) Usually the amount of RNA template is 10pg-100ng as a reference. Since the templates of different species contain different copy numbers of target genes, the templates can be diluted in gradient to determine the optimal amount of template to use.(4) The excitation optical system varies from instrument to instrument, choose to add 50×Low ROX or 50×High ROX according to the instrument using fluorescence quantification.3. Mix well, centrifuge briefly, and collect the solution at the bottom of the tube.4.RT-PCR reaction conditions:Note: 1) The hot start enzyme used in this product must be activated under the condition of pre-denaturation 95℃, 5-10min. 2) It is recommended to use the two-step PCR reaction program, if you can not get good experimental results due to the use of primers with lower Tm value, etc., you can try to carry out the three-step PCR amplification, and the annealing temperature should be set in the range of 56℃-64℃ as a reference... Read More | Product content:M665754Component25 TStorageM665754ATris-HCl, 1 mM, PH 8.01 mL-20℃. Avoid freeze/thaw cycleM665754BE. coli Poly(A) Polymerase, 5 U/µL15 µL-20℃. Avoid freeze/thaw cycleM665754C10×Poly(A) Polymerase Buffer80 µL-20℃. Avoid freeze/thaw Product content:M665754Component25 TStorageM665754ATris-HCl, 1 mM, PH 8.01 mL-20℃. Avoid freeze/thaw cycleM665754BE. coli Poly(A) Polymerase, 5 U/µL15 µL-20℃. Avoid freeze/thaw cycleM665754C10×Poly(A) Polymerase Buffer80 µL-20℃. Avoid freeze/thaw cycleM665754DATP, 10 mM15 µL-20℃. Avoid freeze/thaw cycleM665754ERT Primer, 25 µM90 µL-20℃. Avoid freeze/thaw cycleM665754F5×SuperRT Buffer120 µL-20℃. Avoid freeze/thaw cycleM665754GUltraPure dNTP Mix, 10 mM each30 µL-20℃. Avoid freeze/thaw cycleM665754HSuperRT, 200 U/µL15 µL-20℃. Avoid freeze/thaw cycleM665754IRNase-Free Water1 mL-20℃. Avoid freeze/thaw cycle Product Introduction:This kit uses the method of adding a poly (A) tail at the 3 'end of miRNA to give miRNA a Poly (A) tail, followed by reverse transcription using Oligo (dT) - Universal tag universal reverse transcription primers to synthesize the first stranded cDNA corresponding to miRNA. The miRNA cDNA first strand synthesis kit contains all the reagents required for the miRNA 3 'end Poly (A) tail modification process and the reverse transcription process after modification. This kit has a very high Poly (A) modification and reverse transcription efficiency, which can range from 1 ng-2 µ The first strand of cDNA corresponding to miRNA was effectively obtained from the total RNA of g. And the operation is simple and fast, which can be used to simultaneously detect multiple miRNAs from a synthesized cDNA reaction. This not only reduces errors and saves samples, but also achieves high-throughput detection.Note: This kit must be used in conjunction with the miRNA fluorescence quantitative detection kit.Self prepared experimental materials: 1 ng-2 µ Total RNA of g, or 0.1 ng-1 µ Small molecule RNA of g.Notes:To prevent RNase pollution, attention should be paid to the following aspects:1. Use plastic products and gun heads without RNase to avoid cross contamination.2. Glassware should be dry baked at a high temperature of 180 ℃ for 4 hours before use. Plastic containers can be soaked in 0.5 M NaOH for 10 minutes, thoroughly rinsed with water, and then sterilized under high pressure.3. The solution should be prepared using water without RNase.4. Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.Usage:A. The process of miRNA adding Poly (A) tail:1.based on the amount of RNA used, dilute the total RNA of 10 mM ATP with 1 mM Tris (pH 8.0) according to the following formula: ATP dilution coefficient=5000/__ ngExample: If the initial amount of total RNA is 100 ng, then the ATP dilution coefficient is 5000/100=50. About to dilute ATP 50 times (1 µ 10 mM ATP plus 49 for l µ 1 mM Tris at pH 8.0.2. Add the following reagents to the pre cooled RNase free reaction tube in the ice bath to a total volume of 25 µ L. reagent 25 µlReaction system final concentration total RNA* X µl Up to 2 µg 10×Poly(A) Polymerase Buffer 2.5 µl 1× Diluted ATP in step "1" 1 µl / E. coli Poly(A) Polymerase, 5U/µl 0.5 µl 2.5 U RNase-Free Water up to 25 µl /*The total RNA used in the reaction must contain small molecule RNA.This process can also directly use small molecule RNA (recommended dosage of 2-5) µ L. Please determine the amount added based on the abundance of the target miRNA.3. Gently mix the above reaction solution and briefly centrifuge to collect the liquid at the bottom of the tube. Incubate at 37 ℃ for 15 minutes. After this process is completed, immediately proceed with the synthesis of the first strand cDNA or temporarily store it at -20 ℃. If long-term storage is required, it is recommended to store at -80 ℃.B. The process of synthesizing the first strand of modified miRNA cDNA:1. Add the reagents in the table below to the pre cooled RNase free reaction tube in the ice bath until the final volume reaches 20µl: reagent 20 µlReaction system The above Poly (A) reaction solution 4 µl UltraPure dNTP Mix ，10 mM each 1 µl RT Primer ，25 µM 3 µl 5×SuperRT Buffer 4 µl SuperRT ，200 U/µl 0.5 µl RNase-Free Water 7.5 µl2. Gently mix the above reaction solution and briefly centrifuge to collect the liquid at the bottom of the tube. Incubate at 42 ℃ for 50 minutes.3.85 ℃ for 5 minutes and terminate the reaction. The synthesized cDNA reaction solution can be directly used for fluorescence quantitative detection experiments or stored at -20 ℃ for future use... Read More | Products R669890Component50 TStorageR669890ADNase I1000 U-20℃. Avoid freeze/thaw cycle.R669890B10×Reaction Buffer1mL-20℃. Avoid freeze/thaw cycle.R669890CBuffer RL35 mLRTR669890DBuffer RW140 mLRTR669890EBuffer RW2 (concentrate)11 mLRTR669890FRNase-Free Water10 mLRTR669890GSpin Products R669890Component50 TStorageR669890ADNase I1000 U-20℃. Avoid freeze/thaw cycle.R669890B10×Reaction Buffer1mL-20℃. Avoid freeze/thaw cycle.R669890CBuffer RL35 mLRTR669890DBuffer RW140 mLRTR669890EBuffer RW2 (concentrate)11 mLRTR669890FRNase-Free Water10 mLRTR669890GSpin Columns FL with Collection Tubes50 setsRTR669890HSpin Columns RM with Collection Tubes50 setsRTR669890IRNase-Free Centrifuge Tubes (1.5 mL)100 EART ProductsThis kit adopts centrifugal adsorption columns with high efficiency and specificbinding of nucleic acids and unique buffer system, which can rapidly extract totalRNA from bacteria or cultured animal cells.The reaction can be completed in 30-40minutes, and the extracted total RNA is extremely pure and free of protein and othercontaminants, which is suitable for RT-PCR, Real-Time RT-PCR, microarray analysis,in vitro translation and other experiments. Self-contained reagents: Lysozyme, β-mercaptoethanol, anhydrous ethanol (freshlyopened or for RNA extraction). Pre-experiment Preparation and Important Notes 1. To prevent RNase contamination, attention should be paid to the following aspects:1) Use RNase-free plastics and tips to avoid cross-contamination. 2) RNase-free water should be used to prepare the solution. 3) Operators wear disposable masks and gloves, and change gloves diligently duringthe experiment. 2. Add β-mercaptoethanol to Buffer RL before use to reach a final concentrationof 1%, e.g., add 10 µl of β-mercaptoethanol to 1 ml of Buffer RL. Buffer RL withβ-mercaptoethanol can be stored at 4℃ for 1 month, if precipitation occurs, pleaseheat to dissolve and use.3. Anhydrous ethanol should be added to Buffer RW2 before first use according tothe instructions on the reagent bottle label. 4. All centrifugation steps are carried out at room temperature if not otherwisespecified, and all steps should be performed quickly. Procedure 1. Centrifuge at 12,000 rpm (~13,400 x g) at 4°C for 2 minutes to collect theorganisms (maximum volume of organisms should not exceed 1 x 109) and carefullyremove all supernatants. Note: Supernatants that leave residues can interfere with the subsequent digestionprocess. 2. Thoroughly resuspend the organisms with 100 µl of TE buffer containing Lysozymeand incubate at room temperature. The specific formulation and incubation time areas follows:/The final concentration of Lysozyme in TE bufferincubation timeG-germ400µg/ml3-5minG+germ3mg/ml5-10min 3. Add 350 µl of Buffer RL (check that β-mercaptoethanol has been added beforeuse), vortex and shake to mix (insoluble precipitate may appear in this step), addall of the solution and the precipitate to the filter columns (Spin Columns FL) thathave been loaded into the collection tubes, and centrifuge at 12,000 rpm for 2minutes. 4. Add 250 µl of anhydrous ethanol to the filtrate obtained in the previous stepand mix well (a precipitate may appear at this point). Transfer the resulting solution together with the precipitate to a Spin Columns RM packed in a collectiontube, centrifuge at 12,000 rpm for 1 min, discard the waste solution and put thecolumn back into the collection tube.5. Add 350 µl Buffer RW1 to the adsorbent column, centrifuge at 12,000 rpm for1min, discard the waste liquid and put the adsorbent column back into the collectiontube.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 make a finalvolume of 80µl of reaction solution.7. Add 80µl of DNase I mixture directly to the adsorption column and incubate at20-30°C for 15 minutes.8. Add 350 µl Buffer RW1 to the adsorbent column, centrifuge at 12,000 rpm for1min, discard the waste liquid and put the adsorbent column back into the collectiontube.9. Add 500 µl of Buffer RW2 to the column (check that anhydrous ethanol is addedbefore use), centrifuge at 12,000 rpm for 1 min, and discard the waste solution.10. Repeat step 9.11. Place the adsorbent column back into the collection tube and centrifuge at 12,000rpm for 2 minutes. Note: The purpose of this step is to remove residual ethanol from the adsorptioncolumn; ethanol residue can interfere with subsequent enzymatic reactions (zymography, PCR, etc.).12. Load the adsorption column into a new RNase-Free collection tube, add 30-50 µl of RNase-Free Water to the middle of the adsorption membrane, leave it at roomtemperature for 1 minute, centrifuge at 12,000 rpm for 1 minute, collect the RNAsolution, and store the RNA at -70°C to prevent degradation. Note: 1) The volume of RNase-Free Water should not be less than 30 µl, too smallvolume affects the recovery rate. 2) If you want to increase the RNA yield, repeat step 12 with 30-50 µl of freshRNase-Free Water. If the RNA concentration is to be increased, the resulting solution can be reintroduced into the adsorption column and step 12 repeated... Read More |