| Description | One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 74℃.Application：high-fidelity PCR amplification, point mutation, and gene cloning, etc. The amplified PCR products by Plus DNA polymerase are dA-tailed at 3&#One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 74℃.Application：high-fidelity PCR amplification, point mutation, and gene cloning, etc. The amplified PCR products by Plus DNA polymerase are dA-tailed at 3’ ends and can be directly cloned into T vectors.Definition of activity: One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 74℃.Purity: free of DNA endonuclease and exonuclease, phosphatase, and RNAase.Enzyme storage buffer: 20mM Tris-HCl (pH 7.5), 1mM DTT, 0.1mM EDTA, 100mM KCl, 200µg/ml BSA and 50% (v/v) glycerol.Inactivation or inhibition: Plus DNA polymerase can be deactivated by phenol chloroform extraction. Source：Recombinant hyperthermophilic archaeon Pyrococcus-like DNA polymerase expressed in E. coli.Enzyme storage buffer：20mM Tris-HCl (pH 7.5), 1mM DTT, 0.1mM EDTA, 100mM KCl, 200µg/ml BSA and 50% (v/v) glycerol.Inactivation or inhibition：Plus DNA polymerase can be deactivated by phenol chloroform extraction. Precautions：Because PCR reaction is extremely sensitive, contamination must be avoided during preparation of PCR reactions. Negative control without templates is recommended for all PCR assays to control contamination.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.Instructions for Use：1.Prepare PCR reactions:a.Thaw PCR components at room temperature and mix well prior to use. Keep the Plus DNA polymerase on ice.b.Set up the PCR reaction on ice as follows:To amplify dsDNA 6kbReagentFinal ConcentrationVolumeFinal ConcentrationVolumeNuclease-free water-(36.5-x)µl-(29-y)µl10X Buffer (with Mg2+)1×5µl1×5µldNTP (2.5mM each)0.25mM each5µl0.5mM each10µlTemplate DNA10pg-1µg*xµl10pg-1µg*yµlPrimer mixtures (10µM each)0.2µM each1µl0.4µM each2µl DNA Polymerase2.5U/50µl1µl2.5U/50µl1µlTotal volume-50µl-50µlNote 1: When multiple reactions are required, prepare a master mix including all reagents except template and primer, and then dispense to different nuclease-free PCR tubes. Sometimes, the master mix can also include template and primer.Note 2: When amplifying fragments larger than 6kb, the amount of template should be increased appropriately, but too much template DNA can also easily lead to non-specific PCR amplification products. * The recommended amount of template varies for different types of DNA. In a reaction volume of 50µl, use 100 ng of Mammalian genomic DNA, 100 ng of E. coli genomic DNA, or 5-30 ng of Plasmid DNA. c.Mix the PCR reaction mixture by gentle vortex or pipetting. Centrifuge briefly to collect liquid at the bottom of the PCR tube. d.(Optional) When using a thermocycler without a heated lid, place a drop of mineral oil onto the top of PCR reaction mixture.2.Transfer the PCR reaction to a thermocycler and run thermocycling conditions as follows:StepTo amplify dsDNA 6kbCyclesInitial denaturation94℃ 3min94℃ 3min1Denaturation94℃ 30sec94℃ 30sec30Annealing55℃ 30sec55℃ 30secExtension68℃ 15s/kb68℃ 1min/kbFinal extension68℃ 10min68℃ 15min1Hold4℃ forever4℃ forever-Note 1: The extension temperature can also be set to 72℃, but the amplification will be slightly reduced. For DNA fragments difficult to amplify, denaturation at 92℃ and extension at 72℃ are recommended. Note 2: PCR running conditions should be adjusted based on the template, primer sequence, the length of PCR product or GC content, etc.Note 3: The optimal extension time varies depending on the amplicon length. For amplification of DNA fragments shorter than 6kb, the recommended extension time is 15 seconds per kb (e.g. use 15 seconds to amplify 1kb fragment and 30 seconds to amplify 2kb fragment). When amplifying DNA fragments longer than 6kb, the recommended extension time is 1 minute per kb (e.g., use 5 minutes to amplify 5kb fragment). Note 4: For initial PCR, the number of cycles can be set to 35 to ensure that the expected PCR product can be amplified. The number of cycles for semi-quantitative or quantitative PCR analysis must be optimized appropriately so that the PCR reaction does not reach a plateau.FAQ:1.Few PCR products or no specific bands.a.It could be due to poor design of primers. Use primer design tools for primer design to avoid inappropriate GC content, secondary structure, dimer, annealing temperature, length, specificity and other possible problems. When adding restriction enzyme cutting sites in the primer sequence, the same problems need to be considered. In the case that positive control primers work normally but not your primers, redesign primers.b.DNA to be amplified may have a high GC content. High GC genes are relatively difficult to be amplified. In such a case, GC-rich buffer suitable for amplifying DNA with high GC content can be used, and PCR reaction parameters should be adjusted accordingly. Direct addition of 1-10% DMSO or 5-20% glycerol is also helpful for amplifying fragments with high GC content.c.PCR reactions set up at room temperature tend to produce non-specific bands. It is recommended to set up PCR reactions on ice.d.The presence of secondary structure in primers, primer dimers or short primers, may result in poor annealing of primers to the target sequence. In this case, try touch down or other methods for annealing. A gradual cooling from 65℃ to 55℃ or 50℃ is usually used to make annealing more efficiently. e.The annealing temperature needs to be optimized. If necessary, use a temperature gradient to determine the optimal annealing temperature for each template-primer pair combination. f.Insufficient extension time. The extension time can be extended 2-5 times from the recommended extension time, and can be set to 5 minutes per 1kb for fragments difficult to amplify. g.Insufficient denaturation. To amplify long DNA or high GC DNA fragment, the initial denaturation temperature can be adjusted to 95℃ for 1 min or even 95℃ for 2-4 min.h.Perform PCR reactions on a different thermal cycler to avoid possible problems with the instrument.i.Insufficient number of PCR cycles. Try more PCR cycles, but do not exceed 40 cycles. j.Insufficient amount of template. Add more DNA templates or try nested PCR or secondary PCR. Nested PCR is to design another pair of PCR primers inside the original PCR primers, and then conduct PCR amplification again with the diluted first PCR product as template. Instead, secondary PCR uses the same primers for second PCR amplification with the diluted PCR product as template. Nested PCR usually can remove the non-specific DNA amplification, but secondary PCR cannot.k.DNA sample contains substances that inhibit the PCR reactions. In such a case, template DNA can be purified using appropriate DNA purification methods such as column purification.l.Use high-purity primers.m.Use high-quality dNTP mixture.n.Increase the amount of DNA polymerase appropriately.o.When non-specific products are produced, increase the annealing temperature appropriately.p.Positive and negative controls are always recommended when optimizing PCR reactions.2.Occurrence of non-specific bands or DNA smear when examined by agarose gel electrophoresis.a.Increase the annealing temperature by 2-5℃.b.Reduce the amount of DNA template.c.PCR reactions set up at room temperature tend to produce non-specific bands. It is recommended to set up PCR reactions on ice.d.Reduce the amount of Plus DNA polymerase appropriately.e.Reduce the extension time appropriately... Read More | Product introduction：Aladdin ® SE is a kind of fluorescent dye with amino reactive activity. The SE group of these dyes can react with the amino group to produce a stable amide bond. Compared with other similar dyes on the market, aladdin ® is a new generation of fluorescent dyes Product introduction：Aladdin ® SE is a kind of fluorescent dye with amino reactive activity. The SE group of these dyes can react with the amino group to produce a stable amide bond. Compared with other similar dyes on the market, aladdin ® is a new generation of fluorescent dyes with stronger stability, better water solubility and better fluorescence intensity. Product parameters： Absmax/Em(nm)：648/664；Absmax/Em(nm)：0.03；Extinction coefficient(ε)：240000；Optimal DOL(IgG)：3-6； Usage:1. Experimental materials(1) IgG: IgG must not contain amine chemicals that can react with dyes, such as amino acids, Tris, BSA, gelatin, etc. If IgG contains such chemicals, PBS buffer with pH~7.4 should be used for pre dialysis treatment. The presence of azide compounds does not affect the labeling reaction.(2) Anhydrous DMSO(3) NaHCO3(4) Sephadex gel G-25 dialysis column(5) PBS buffer (pH~7.4)(6) NaN3(7) BSA2. Marking methods and steps(1) Prepare to label antibodiesDilute the antibody with 0.1 M NaHCO3 solution (pH~8.3) to a final concentration of 2.5 mg/mL. If the product is pre diluted with phosphate buffer, such as PBS buffer (without amino compounds), approximately 1/10 volume of 1M NaHCO3 mother liquor can be directly added to the buffer to achieve a final NaHCO3 concentration of 0.1 M.Note: When the protein concentration is 2.5 mg/mL, the labeling efficiency is approximately 35%. Protein concentrations below 2.5 mg/mL can also be used for labeling, but the labeling efficiency will decrease. When the protein concentration is higher than 5 mg/mL, the labeling efficiency may be higher. Due to differences in buffer and protein purity, more precise labeling efficiency is determined by practical operating conditions. If the protein concentration is too low, it can be concentrated by ultrafiltration.(2) Prepare dye storage solutionPreheat one tube at room temperature µ YF of Mole ® SE, add 0.1 mL of anhydrous DMSO to the tube, thoroughly vortex dissolve the dye, and prepare a dye storage solution with a concentration of 10 mM. If a trace amount of protein is used for labeling reactions, the dye needs to be diluted to a lower concentration.Note: a The remaining dye storage solution should be stored at a low temperature of -20 ℃ for future use. If anhydrous DMSO is used to prepare dye storage solution, the dye can be stored for at least one month.b. Dyes can also be prepared with deionized water, but due to the slow hydrolysis of dyes in water, it is best to prepare water based storage solutions for immediate use.(3) Mark reaction stepsa. Stir or vortex the protein solution, gradually adding 15-25 drops µ L dye storage solution (10 mM), with a molar ratio of dye/protein in the range of 9:1 to 15:1. YF ® Please refer to the table above for the range of DOL (number of dyes bound to each protein molecule) for SE labeled IgG antibodies.b. Stir the reaction at room temperature for 1 hour, and for trace labeling, shake and incubate on a shaker for 1 hour.Note: At the same time of the binding reaction, proceed to step 2 (4) to balance the dextran gel G-25 dialysis column.(4) Isolation of marker proteins from reaction solutiona. PBS buffer (pH~7.4) was used to balance the dextran gel G-25 dialysis column (10 mm × 300 mm).b. Add the reaction solution from step 3 (b) to the column and elute with 1 x PBS buffer.The first washed out chromophore is a dye protein complex.Note: a For small-scale labeling reactions, in order to avoid excessive dilution of the product, ultrafiltration devices can be used to remove free dyes from the complex.b. After the binding reaction is completed, if the dye protein complex is not separated in time, 50 can be added µ Terminate the reaction with L 1M lysine. In most cases, this operation is not necessary because the remaining unreacted dyes have been fully hydrolyzed at the end of the reaction.3. Determine DOL(1) The determination of protein concentration and antibody concentration can be calculated using the following formula:C (mg/mL)={[A280- (Amax x x Cf)]/1.4} x dilution factor;a. C refers to the concentration of antibodies collected in the experiment;b. Dilution factor refers to the dilution factor used in photometric measurements;c. A280 and Amax refer to the absorbance at 280 nm and the absorbance at the absorption wavelength, respectively;d. Cf is the correction factor, YF ® Please refer to the table above for the Cf value of SE dyes;Note: The protein solution eluted through the column may have a high concentration when used directly for absorbance detection, so it needs to be diluted to approximately 0.1 mg/mL. The dilution factor (i.e. dilution factor) needs to be determined from the initial number of antibodies (e.g. 5 mg) and the overall elution of protein solutionEstimate based on the product.(2) Estimation of DOLDOL is calculated using the following equation:DOL=(Amax x x Mwt x Dilution Factor)/（ ε X C)a. Amax, dilution factor, C value has been clearly defined in 3 (1);b. Mwt refers to the molecular weight of IgG (150000);C. c ε It's YF ® The molar absorption coefficient of SE, refer to the table on the first page;d. Mark YF ® The optimal DOL value for SE IgG antibodies can be found in the table on the first page. Although DOL values may fluctuate, good experimental results can also be achieved.Matters needing attention：1. if the labeled protein needs long-term storage, it is recommended to add 5-10 mg/ml BSA and 0.01-0.03% NaN3 to prevent protein denaturation and microbial breeding. Store at 4 ℃ away from light. If glycerol with a final concentration of 50% is added, it can be stored at -20 ℃. It can be stably stored for more than one year. 2. keep away from light during operation. The mixing speed should be appropriate to avoid bubbles. 3. when installing the chromatographic column, try to make the column body uniform, the column surface flat, and free of bubbles and cracks. 4. pay attention to adding the sample when the column top buffer is tangent to the gel plane. When eluting, add the eluent when the sample is tangent to the gel plane. 5. other factors affecting the labeling efficiency also include temperature, reaction time, pH, the amount of fluorescent dye and protein, etc., which should be controlled. 6. for your safety and health, please wear laboratory clothes and disposable gloves.Scope of application：Protein nucleic acid labeling dye... Read More | Acid phosphatase is an esterase with broad activity at an optimal pH below 7.0. There are three isozymes, EI, EII, and EIII of similar molecular weight (55 kDa± 5 kDa). Their optimum pH's are 5.5, 4.5, and 4.0 respectively. Acid phosphatase activity was observed by Teller Aladdin Library Acid phosphatase is an esterase with broad activity at an optimal pH below 7.0. There are three isozymes, EI, EII, and EIII of similar molecular weight (55 kDa± 5 kDa). Their optimum pH's are 5.5, 4.5, and 4.0 respectively. Acid phosphatase activity was observed by Teller Aladdin Library Archives in 1954 in preparations of a wheat germ lipase described by Singer JBC, 174, 11, in 1948. Equivalent commercial preparations have been distributed labeled as lipase and acid phosphatase thus generating some confusion. Subsequent work has confirmed that the non-specific esterase activity of the wheat germ preparation may be measured both as lipase (triacetin as substrate) and phosphatase. The enzyme assay is based on the work of Brandenberger and Hanson (Helv. Chim. Acta, 36, 900, 1953) and Hofstee ( Arch. Biochem. Biophys., 51, 239, 1954).Acid phosphatase (APase) non-specifically catalyzes the hydrolysis of monoesters and anhydrides of phosphoric acid to produce inorganic phosphate. It is used to study the production, transport, and recycling of phosphate and the metabolic and energy transduction processes of the cell.Characteristics of Acid Phosphatase from Wheat Germ:Molecular weight: 55,000 ± 5,000 (Verjee 1969).Composition: Three isozymes of closely similar molecular weights have been reported by Verjee (1969): EI, EII, and EIII. See also Brouillard and Ouellet (1965).Optimal pH: EI - 5.5, EII - 4.5, and EIII - 4.0. (Verjee 1969).Specificity: The enzyme has a broad esterase activity. See Joyce and Grisolia (1960). It shows highest activity for pyrophosphate.Inhibitors: Fluoride, molybdate and orthophosphate (Verjee 1969)... Read More | Purity:>90%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description: High-mobility group box 1 protein (HMGB1), also known as HMG-1 or amphoterin previously, is a member of the HMGB family consisting of three members, HMGB1, HMGB2, and HMGB3. HMGB1 is a DNA-binding nuclear protein,Purity:>90%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description: High-mobility group box 1 protein (HMGB1), also known as HMG-1 or amphoterin previously, is a member of the HMGB family consisting of three members, HMGB1, HMGB2, and HMGB3. HMGB1 is a DNA-binding nuclear protein, released actively following cytokine stimulation as well as passively during cell death. It is the prototypic damage-associated molecular pattern (DAMP) molecule and has been implicated in several inflammatory disorders. HMGB1 signals via the receptor for advanced glycation end-product (RAGE) and members of the toll-like receptor (TLR) family. The most prominent HMGB1 protein and mRNA expression arthritis are present in pannus regions, where synovial tissue invades articular cartilage and bone. HMGB1 promotes the activity of proteolytic enzymes, and osteoclasts need HMGB1 for functional maturation. As a non-histone nuclear protein, HMGB1 has a dual function. Inside the cell, HMGB1 binds DNA, regulating transcription, and determining chromosomal architecture. Outside the cell, HMGB1 can serve as an alarmin to activate the innate system and mediate a wide range of physiological and pathological responses. Extracellular HMGB1 represents an optimal " necrotic marker" selected by the innate immune system to recognize tissue damage and initiate reparative responses. However, extracellular HMGB1 also acts as a potent pro-inflammatory cytokine that contributes to the pathogenesis of diverse inflammatory and infectious disorders. HMGB1 has been successfully therapeutically targeted in multiple preclinical models of infectious and sterile diseases including arthritis. As shown in studies on patients as well as animal models, HMGB1 can play an important role in the pathogenesis of the rheumatic disease, including rheumatoid arthritis, systemic lupus erythematosus, and polymyositis among others. Besides, enhanced postmyocardial infarction remodeling in type 1 diabetes mellitus was partially mediated by HMGB1 activation... Read More | Format:10X ConcentrateProtein:Casein |