| Description | 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 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 | Products contentN665730Component24 T96 TStorageN665730ATPS V50 144 µL576 µL-20℃. Avoid freeze/thaw cycle.N665730B5×FA Reaction Buffer144 µL576 µL-20℃. Avoid freeze/thaw cycle.N665730C2×HiFidelity PCR Mix600 µL2×1.2 mL-20℃. Avoid freeze/thaw Products contentN665730Component24 T96 TStorageN665730ATPS V50 144 µL576 µL-20℃. Avoid freeze/thaw cycle.N665730B5×FA Reaction Buffer144 µL576 µL-20℃. Avoid freeze/thaw cycle.N665730C2×HiFidelity PCR Mix600 µL2×1.2 mL-20℃. Avoid freeze/thaw cycle.N665730DPPM48 µL192 µL-20℃. Avoid freeze/thaw cycle.* This kit is suitable for human genomic DNA library construction with a starting template DNA input of 50 ng. We also have transposase library construction kits for human genomic DNA starting at 5 ng and 1 ng, so it is recommended to use different kits for different starting amounts of DNA in order to obtain higher quality libraries. Products IntroductionThis kit is developed for Illumina's high-throughput sequencing platform and provides the enzyme premix system and reaction buffer for genomic DNA library construction, including all components except PCR primers. Compared with the traditional library construction kits, this kit adopts the new transposase method for library construction, which can complete DNA fragmentation, end repair and junction reaction in one simple enzymatic reaction, significantly reducing the amount of template, reducing the number of experimental steps, and shortening the time of library construction; it adopts the high-fidelity DNA polymerase for library enrichment, and the preference-free PCR amplification can expand the coverage area of the sequence, which can be used for efficient and effective sequencing. The use of high-fidelity DNA polymerase for library enrichment and preference-free PCR amplification broadens the coverage area of the sequence and enables efficient preparation of DNA libraries for Illumina's second-generation sequencing platform. The kit is suitable for DNA libraries with a starting template of 50 ng, and all reagents in the kit have been subjected to strict quality control and functional validation to maximize the stability and reproducibility of library construction. Product Features ● DNA fragmentation and junction ligation in one step.● Ultra-fidelity amplification minimizes amplification-preferred steps.Provide your own instruments, kits and consumables1. Magnetic frame: DynaMagTM-2 is recommended.2. DNA purification and recovery kit: It is recommended to use DNA purification and recovery kit by magnetic bead method.3. Library PCR primer kits: transposase method for second-generation sequencing multi-sample primer kits are recommended. 4. Anhydrous ethanol, deionized water (pH between 7.0 and 8.0).5. Reaction tubes: It is recommended to use low adsorption PCR tubes and 1.5 ml centrifuge tubes. Tips: It is recommended to use high quality filter tips to prevent contamination of kits and library samples. Pre-experiment Preparation and Important Notes1. Avoid repeated freezing and thawing of reagents.2. PCR products are easily contaminated due to improper operation, resulting in inaccurate results. It is recommended to isolate the PCR reaction system preparation area from the PCR product purification area, and to use special pipettes to clean the experimental areas at regular intervals.3. Bead purification: the beads should be equilibrated to room temperature before use, all operations on the beads should be carried out at room temperature, 80% ethanol should be dispensed freshly, the beads should be rinsed and dried until the surface is free of liquid reflections and has a frosted appearance, insufficient drying of the beads will cause ethanol residue that will affect the subsequent experiments, and over-drying of the beads will affect the efficiency of DNA recovery.4. The kit is suitable for human genomic DNA library construction, if the DNA sample is a PCR product, it should be ensured that its length>.500 bp, since transposases do not work on DNA ends, it is recommended to extend the PCR product by 50-100 bp at each end of the PCR product to avoid low coverage of the ends for sequencing.Sample PreparationDNA purity requirements: A260/A280 = 1.8-2.0. Sample DNA: dissolve in ultrapure water. DNA Quantification: Too much or too little DNA will affect the quality of the library. It is recommended to use Nano to test the purity of the genomic DNA and then use Qubit to test the concentration of the genome (do not use any absorbance-based assay for template quantification).Schematic diagram of DNA banking processprocedureDNA fragmentation, junction reaction1. Add the following reagents to a 200 µl PCR tube: 2. Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.3. Place the above PCR tubes in the PCR instrument with the hot cap on and program the reaction as follows:DNA should be purified immediately after the fragmentation reaction has been performed and the transposase is still in a high state of activity.to prevent smaller library fragments due to DNA over-fragmentation. Purification of fragmentation productsWe recommend the use of the Century Magnetic Bead Method DNA Purification and Recovery Kit.1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.2. Add 50 µl of magnetic beads equilibrated to room temperature to the fragmentation product, vortex and shake for 5 seconds, then let stand at room temperature for 5 minutes.3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant solution until the solution is clear (approximately 3-5 minutes), carefully aspirate the supernatant and discard, avoiding contact with the beads that have bound the target DNA. Note: Do not discard the beads.4. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 µl of freshly prepared 80% ethanol to the centrifuge tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.Note: When adding ethanol, the liquid must not be blown directly onto the beads.5. Repeat step 4.6. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, then add 23 µlddH2O to solubilize.Note: Do not over-dry the beads as this may affect the elution efficiency.7. Remove the tube from the magnetic rack, vortex to completely resuspend the beads, and allow to stand at room temperature for 5 minutes. Centrifuge briefly, place the tube on the magnetic rack until the solution is clear, and transfer 21 µl of supernatant to a new 200 µl PCR tube.PCR amplification Add the following reagents to the 200 µl PCR tube: Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.3. Place the above PCR tubes in the PCR instrument with the hot cap on and program the reaction as follows:Selective recovery of library DNA fragmentsIt is recommended to use CombiVision Magnetic Beads DNA Purification and Recovery Kit for selective recovery of DNA fragments. When different sizes of DNA fragments are required, the amount of magnetic beads to be used is different, please refer to the attached table for the specific amount of magnetic beads to be used (if other brands of magnetic beads are used, you need to find out the optimal amount of magnetic beads to be used on your own).Note: Amplification products can also be fragment length sorted and purified using the Gum Recovery Kit. If there is no special requirement for library length distribution, the amplification products can also be purified without selective recovery of DNA fragments as described on page 6 of the manual.1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.2. Transfer the PCR product to a 1.5 ml centrifuge tube, rehydrate to 100 µl and add several volumes of magnetic beads equilibrated to room temperature, vortex for 5 seconds and let stand at room temperature for 5 minutes.3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant until the solution is clear, and carefully aspirate the supernatant and transfer it to a new 1.5 ml centrifuge tube.Note: Do not discard the top clear.4. Add several volumes of magnetic beads to the supernatant, vortex and shake for 5 seconds, then let stand at room temperature for 5 minutes.5. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant until the solution is clear, carefully aspirate the supernatant and discard it, avoiding contact with the beads that have bound the target DNA.Note: Do not discard the beads.6. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 µl of freshly prepared 80% ethanol to the tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.Note: When adding ethanol, the liquid must not be blown directly onto the beads.7. Repeat step 6 once.8. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, add 20 µl of ddH2O to solubilize.Note: Do not over-dry the beads as this may affect the elution efficiency.9. Remove the centrifuge tube from the magnetic rack, vortex and oscillate to completely resuspend the beads, and let stand at room temperature for 5 minutes. Leave brieflycentrifuge, place the tube on a magnetic rack until the solution is clear, and transfer the supernatant solution to a new centrifuge tube. Table: Suggested amount of magnetic beads for different segment selection recoveryLibrary DNA fragment purificationWe recommend the use of the Century Magnetic Bead Method DNA Purification and Recovery Kit.1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.2. 50 µl of magnetic beads equilibrated to room temperature were added to the PCR product, vortexed and shaken for 5 seconds, and then left to stand at room temperature for 5 minutes.3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant solution until the solution is clear (approximately 3-5 minutes), carefully aspirate the supernatant and discard it, avoiding contact with the beads that have bound the target DNA. Note: Do not discard the beads.4. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 µl of freshly prepared 80% ethanol to the centrifuge tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.Note: When adding ethanol, the liquid must not be blown directly onto the beads.5. Repeat step 4.6. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, add 25 µl of ddH2O to solubilize.Note: Do not over-dry the beads as this may affect the elution efficiency.7. Remove the tube from the magnetic rack, vortex to completely resuspend the beads, and allow to stand at room temperature for 5 minutes. Centrifuge briefly, place the tube on the magnetic rack until the solution is clear, and transfer the supernatant solution to a new tube.Library quality controlDetermination of library concentrationIn order to obtain high-quality sequencing results, accurate quantification of DNA libraries is required, and the first recommendation is to use Real-timePCR methods are used for absolute quantification of DNA libraries. Additionally, fluorescent dye methods such as the Qubit method or the fluorescent dye picogreen method can be used; do not use quantification methods based on absorbance measurements here. The following approximate formula can be used to convert the molar concentration of the DNA library. Average total length of librariesApproximate conversion formula Library fragment distributionThe prepared DNA libraries can be detected by agarose gel electrophoresis or Agilent 2100 Bioanalyzer.Range of segment length distributions... Read More | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | Inquire |