| Description | Biotin Ligase (also known as BirA, EC 6.3.4.15) can activate biotin to form biotinyl-5'-adenylate, and specifically transfer biotin to biotin receptor proteins (such as AviTag fusion proteins), thereby biotinylating the proteins.Component ListB1505408Component20µg2×20µBiotin Ligase (also known as BirA, EC 6.3.4.15) can activate biotin to form biotinyl-5'-adenylate, and specifically transfer biotin to biotin receptor proteins (such as AviTag fusion proteins), thereby biotinylating the proteins.Component ListB1505408Component20µg2×20µgStorageB1505408ABiotin-Protein Ligase (1mg/ml)20µl2×20µl-80℃. Avoid freeze/thaw cycle.B1505408B10×Biotin Ligase Buffer A500µl1 mL-80℃. Avoid freeze/thaw cycle.B1505408C10×Biotin Ligase Buffer B500µl1 mL-80℃. Avoid freeze/thaw cycle.Activity and Purity The biological activity of Biotin-Protein Ligase is ≥ 7,500 Units/µg. Purity is > 95%, with no detectable protease activity.Storage Conditions1. Shipped on dry ice. After thawing, aliquot the product and avoid repeated freeze-thaw cycles.2. For long-term storage of Biotin-Protein Ligase, store at -80°C; for short-term use, store at -20°C; for frequent use, it can be stored at 4°C for a short period. When stored at 4°C, the enzyme can retain > 90% of its activity for three months.3. Store Ligase Buffer A and Ligase Buffer B at -20°C; repeated freeze-thaw cycles are acceptable.Example of Reaction SystemReactant ComponentVolume10×Biotin Ligase Buffer A2.5 µl10×Biotin Ligase Buffer B2.5 µlBiotin Ligase0.17 µlProtein (polypeptide) substrate1 - 5 µgdH₂Oup to 25 µl Total volume is 25 µl. Incubate at 25°C for 12 - 16 hours to fully biotinylate the substrate.Precautions1. The substrate of this product is AviTag fusion protein.2. If the substrate protein (polypeptide) needs to be stored at a lower temperature, the reaction temperature can be appropriately reduced and the enzymatic reaction time should be prolonged.3. Many common buffer components such as sodium chloride (>100 mM), glycerol (>5% W/V), and ammonium sulfate (>50 mM) can inhibit the activity of biotin ligase. When these reagents are necessary for the substrate protein (polypeptide), their concentrations should be reduced as much as possible.4. The substrate can contain Tris (pH 8.0), with an optimal concentration of 10 mM and no more than 50 mM.FAQsQ: How to prevent proteases from degrading the substrate?A: The biotin ligase of this product undergoes strict quality control and has no protease activity. If the substrate protein used for biotinylation is unpurified crude protein, it is recommended to add an appropriate amount of protease inhibitors to prevent degradation during the biotinylation process.Q: How to determine the optimal amount of this enzyme and reaction conditions for the reaction?A: Due to the great differences in the properties of different proteins and the certain uncertainty in biological enzyme reactions, the reaction conditions described in the manual are not completely applicable to all proteins. It is recommended that users conduct a pre-experiment: first take a small amount of protein, divide it into several equal amounts of substrates, add enzymes with different dilutions, corresponding amounts of Buffer A and B, react at 30°C for 1 h (or other time and temperature conditions), terminate the reaction with SDS-PAGE Loading Buffer, perform electrophoresis and transfer to an NC membrane for Western Blotting detection (after blocking with BSA or skim milk powder, directly incubate with commercially available HRP-Streptavidin, and develop color with DAB), compare the biotinylation situation, and select the optimal conditions. Since the binding force between Streptavidin and Biotin is extremely strong, only a small amount of biotinylated protein is needed for observation results when used for Western Blotting detection.Q: What should I do if the concentration of my substrate protein is quite low and it is not easy to concentrate during biotinylation?A: Add more enzyme and appropriately prolong the reaction time when conducting the enzymatic reaction with the same amount of substrate. However, there is a problem that cannot be ignored: the enzyme is a protein, and when the amount of enzyme increases, the amount of protein introduced into the system also increases. If subsequent experiments do not allow the presence of a large amount of heterologous proteins, then prolonging the reaction time will be the only choice.Q: Does detergent have any effect on the biotinylation reaction?A: Through experimental detection, 0.2% Tween 20 has no effect on the reaction. This product is only for experimental scientific research use. If any unit or individual uses this product for other special purposes specifically stipulated by the state such as clinical diagnosis and treatment, our company will not bear any responsibility... Read More | Inquire | Product content: M665794Component125 TStorageM665794A2×miRNA qPCR Mixture (ROX)2×750 µL-20℃. Avoid freeze/thaw cycleM665794BReverse Primer, 10 µM60 µL-20℃. Avoid freeze/thaw cycleM665794CddH2O1.5 mL-20℃. Avoid freeze/thaw cycle Product Introduction:This kitProduct content: M665794Component125 TStorageM665794A2×miRNA qPCR Mixture (ROX)2×750 µL-20℃. Avoid freeze/thaw cycleM665794BReverse Primer, 10 µM60 µL-20℃. Avoid freeze/thaw cycleM665794CddH2O1.5 mL-20℃. Avoid freeze/thaw cycle Product Introduction:This kit uses the principle of SYBR Green I chimeric fluorescent dye method for miRNA fluorescence quantitative PCR detection. The kit includes 2 x miRNA qPCR Mixture and Reverse Primer required for detection. 2 x miRNA qPCR Mixture is a new generation pre mixed form of fluorescence quantitative PCR detection reagent specially developed for miRNA quantitative detection. The fluorescent dye SYBR Green I contained in it can bind to all double stranded DNA, making the product suitable for detecting different target sequences without the need to synthesize specific labeled probes. The GoldStar Taq DNA polymerase is a chemically modified and highly efficient thermal starter enzyme, coupled with a unique buffer system, which enhances reaction specificity, sensitivity, and enables accurate quantification of miRNA over a wider range. The 2x miRNA qPCR Mixture contains ROX dye and is suitable for fluorescence quantitative PCR instruments that require ROX as a calibration dye.Note: This kit must be used in conjunction with the miRNA cDNA first strand synthesis kit.Self prepared experimental materials: qPCR upstream primer.Forward Primer design principles:1. Follow the most common principles of primer design.2.Based on mature miRNA sequences, replacing U with T is the most basic and simplest design method.3.The Tm value of the downstream primer provided in the reagent kit is 63.6 ℃, and the Tm value of the upstream primer should be designed to be around 63.6 ℃ as much as possible.4. If the Tm value of the primer directly designed according to principle "2" is too low, several bases (preferably G or C bases) can be added to the 5 'end of the primer; One or several A bases can also be added at the 3 'end; Alternatively, both the 5 'and 3' ends can be modified simultaneously.5.If the Tm value of a primer designed directly according to principle "2" is too high, several bases can be removed from the 5 'or 3' end of the primer.Notes:1. Before using the reagent, please gently mix it upside down to avoid foaming, and use it after a brief centrifugation.2. The amount of miRNA first strand cDNA added should not exceed 10% of the volume of Real time PCR.3. For special detection systems, high content of cDNA templates can easily lead to non-specific amplification. Dilute cDNA appropriately (10 or 100 times dilution) based on the abundance of detected miRNAs.4. The 2x miRNA qPCR Mixture in this product contains SYBR Green I and ROX dyes. When storing this product or preparing PCR reaction solution, strong light exposure should be avoided.5. Avoid repeated freezing and thawing of this product. Repeated freezing and thawing may cause a decrease in product performance. This product can be stored at -20 ℃ for long-term storage. If frequent use is required in the short term, the 2xmiRNA qPCR Mixture can be stored at 2-8 ℃. However, the Reverse primer still needs to be stored at -20 ℃.Operation steps:1. Melt 2 x miRNA qPCR Mixture and Reverse Primer at room temperature (10 µ M). 2. When using, please gently mix the 2x miRNA qPCR Mixture upside down to avoid foaming, and use after brief centrifugation. If the reagent is not well mixed, its reaction performance will decrease.3. Place the reagent on ice and prepare the reaction system according to the following table: reagent volume final concentration 2×miRNA qPCR Mixture（ROX） 10 µl 1× Forward primer（10 µM） 0.4µl 0.2 µM Reverse primer（10 µM） 0.4µl 0.2 µM MiRNA first strand cDNA X µl — ddH2O up to 20 µl —4. The reaction program is set as follows:Attention!The pre denaturation reaction of this product must be completed at 95 ℃ for 10 minutes! Note: 1) The hot start enzyme used in this product must be activated under pre denaturation conditions of 95 ℃ and 10 minutes.2) The annealing temperature should be set at 60-64 ℃ as a reference range. When non-specific reactions occur, the annealing temperature can be increased... Read More | Products contentN665993Component240 TStorageN665993AIndex N501 Primers for Illumina240 µL-20℃. Avoid freeze/ Thaw cycle.N665993BIndex N925-N948 Primers for Illumina24×10 µL-20℃. Avoid freeze/ Thaw cycle. Products Introduction This kit is a companion kit to the transposase-Products contentN665993Component240 TStorageN665993AIndex N501 Primers for Illumina240 µL-20℃. Avoid freeze/ Thaw cycle.N665993BIndex N925-N948 Primers for Illumina24×10 µL-20℃. Avoid freeze/ Thaw cycle. Products Introduction This kit is a companion kit to the transposase-based Rapid DNA Library Construction Kit for Illumina platform library construction. Each kit contains one N5 primer and 24 N7 primers, which can be used to prepare 24 different single-ended Index libraries. All reagents provided in the kits have been subjected to stringent quality control and functional validation to maximize the stability and reproducibility of library construction. The libraries can be used for sequencing on Illumina platforms such as HiSeq X-10/4000/2500/2000 and MiSeq. Provide your own instruments, reagents and consumables1. Magnetic frame: DynaMagTM-2 is recommended.2. DNA purification and recovery kit: It is recommended to use Kangwei DNA purification and recovery kit by magnetic bead method.3. DNA building kit: It is recommended to use the Kangwei Century transposase method second-generation sequencing rapid DNA building kit.4. Anhydrous ethanol.5. Reaction tubes: It is recommended to use low adsorption PCR tubes with 1.5 ml centrifuge tubes;Tip: It is recommended to use a high quality filter tip to prevent contamination of kits and library samples. Pre-experiment Preparation and Important NotesPlease centrifuge briefly before opening the cap so that the liquid collects at the bottom of the tube to avoid cross-contamination between different primers. ProcedureFor the use of the CombiVision Second Generation Sequencing Multisample Primer Kit, please follow the CombiVision Second Generation Sequencing Rapid DNA Library Kit protocol.Index N501 Primer for IlluminaIndex N901-N996 Primer for Illumina... Read More | Products contentNote: The amount of individual primers used is 1 µl, each N7-end primer can perform 10 DNA library constructs, and each kit can perform 240 DNA library constructs.Products IntroductionThis kit is a companion kit to the transposase-based Rapid DNA Library Construction Kit for Products contentNote: The amount of individual primers used is 1 µl, each N7-end primer can perform 10 DNA library constructs, and each kit can perform 240 DNA library constructs.Products IntroductionThis kit is a companion kit to the transposase-based Rapid DNA Library Construction Kit for Illumina platform library construction. Each kit contains one N5 primer and 24 N7 primers, which can be used to prepare 24 different single-ended Index libraries. All reagents provided in the kits have been subjected to stringent quality control and functional validation to maximize the stability and reproducibility of library construction. The libraries can be used for sequencing on Illumina platforms such as HiSeq X-10/4000/2500/2000 and MiSeq. Provide your own instruments, reagents and consumables1. Magnetic frame: DynaMagTM-2 is recommended.2. DNA purification and recovery kit: It is recommended to use Kangwei DNA purification and recovery kit by magnetic bead method.3. DNA building kit: It is recommended to use the Kangwei Century transposase method second-generation sequencing rapid DNA building kit.4. Anhydrous ethanol.5. Reaction tubes: It is recommended to use low adsorption PCR tubes with 1.5 ml centrifuge tubes; Tip: It is recommended to use a high quality filter tip to prevent contamination of kits and library samples. Pre-experiment Preparation and Important NotesPlease centrifuge briefly before opening the cap so that the liquid collects at the bottom of the tube to avoid cross-contamination between different primers. procedureFor the use of the CombiVision Second Generation Sequencing Multisample Primer Kit, please follow the CombiVision Second Generation Sequencing Rapid DNA Library Kit protocol. Index N501 Primer for Illumina Index N901-N996 Primer for Illumina... Read More |