| Description | Products contentProducts IntroductionThe 2.5×TFS Master Mixture is a premixed system for all types of multiplexed PCRs. It contains DNA polymerase, PCR Buffer, dNTPs, Mg2+, stabilizers and enhancers at a concentration of 2.5×, which makes it easy and fast to use.The DNA polymerase Products contentProducts IntroductionThe 2.5×TFS Master Mixture is a premixed system for all types of multiplexed PCRs. It contains DNA polymerase, PCR Buffer, dNTPs, Mg2+, stabilizers and enhancers at a concentration of 2.5×, which makes it easy and fast to use.The DNA polymerase contained in the 2.5×TFS Master Mixture is a genetically engineered recombinant enzyme with 5'→3' DNA polymerase activity and no 5'→3' exonuclease activity; it is a new antibody-modified hot-start enzyme that can effectively reduce non-specific amplification caused by non-specific binding of primer and template or primer dimerization at room temperature. The DNA polymerase is modified by a new type of antibody, which is an antibody-modified hot-start enzyme, and can effectively reduce the non-specific amplification caused by the non-specific binding of primers and templates or primer dimerization at room temperature, and at the same time, it has the excellent features of short activation time, strong amplification ability, high sensitivity, good stability, etc. The unique PCR buffer system and the hot-start enzyme are suitable for the PCR. The unique combination of PCR buffer system and hot starter enzyme significantly improves the PCR amplification efficiency, sensitivity and inhibitor tolerance.The product has a wide range of applications, not only for general and dye-based real-time fluorescence PCR, but also for forensic multiple STR amplification reaction, which can be used in forensic analysis, parentage identification and scientific research and other human genetic identification. caveat1. Before use, please mix the product gently by turning it up and down after it is completely melted and centrifuged briefly.2. Avoid repeated freezing and thawing of the product, which may degrade its performance. This product can be stored at -20℃ for a long time. UsageThe following examples are STR reaction systems and conditions, which should be improved and optimized according to the specific use, template, primer structure, target fragment size and amplification effect.PCR reaction systemExtract the DNA amplification reaction system:Blood Card Direct Expansion Reaction System:Attention:1) When designing the primers, the difference between the Tm of each primer should be minimized, and the difference should be controlled within 5℃ as far as possible. If the amplification efficiency is not high, the concentration of primers can be increased; if non-specific amplification occurs, the concentration of primers can be decreased to optimize the reaction system. For optimal amplification, it is recommended that the primer mixture be vortexed for 10 s and centrifuged briefly before use.(2) The amount of DNA template is usually 0.1 ng-1 ng of human genomic DNA as a reference, and the amount of template input can be adjusted according to the amplification effect to determine the optimal amount of template to use.3) Human genome contamination should be avoided during the operation, and a set of negative control (no DNA) is recommended for the experiment.2. PCR reaction conditionsAttention:1) Two-step PCR reaction program is recommended. If you can not get good results due to low Tm value of the primers or large difference in Tm value between primers, you can try to use three-step PCR amplification, the annealing temperature should be set in the range of 55℃-65℃ as a reference (the annealing temperature is usually 5℃ lower than the Tm value), and the extension temperature should be set at 72℃.(2) When good amplification results are not obtained, the annealing and extension time can be appropriately prolonged to 120 s-150 s. The annealing and extension time can be extended to 120 s-150 s. (3) When the PCR product detection appears to be incomplete plus A, the final extension time can be appropriately extended to 30-40 min. 4) The number of cycles can be set according to the downstream application of the amplified product, if the number of cycles is too small, the amplification is insufficient, the recommended number of cycles is 28-31 cycles.5) Blood card direct amplification can be based on the actual amplification effect to increase the 72 ℃ lysis step to improve the amplification efficiency.6) When using the ABI 9700 Thermal Cycler, perform amplification in MAX mode.7) PCR products can be stored at 2-8°C for short-term storage or at -20°C for long-term storage.Attention:(1) Usually, a primer concentration of 0.2 M can give better results, and 0.1-1.0 µM can be used as a reference for setting the range. If the amplification efficiency is not high, the concentration of primer can be increased; if non-specific reaction occurs, the concentration of primer can be decreased to optimize the reaction system.(2) The final 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 manual of the instrument or the specific requirements for the use of each fluorescent probe to adjust the concentration.3) Usually the amount of DNA template is 10-100 ng of genomic DNA or 1-10 ng of cDNA as a reference, as templates of different speciesThe number of copies of the target gene contained in them varies, and a gradient dilution of the template can be performed to determine the optimal amount of template to use. PCR reaction conditions :(1) The enzyme used in this product is activated by pre-denaturation at 95°C for 30 s. Most of the templates can be deconvoluted well under this condition. Under this condition, most of the templates can be well unchained. For templates with high GC content and complex secondary structure, the pre-denaturation time can be extended to 1 minute, so that the starting template can be fully unchained, and if the high temperature treatment time is too long, it will affect the activity of the enzyme; for simple templates, pre-denaturation can also be used for 20 s, and the optimal pre-denaturation time can be determined according to the template situation.(2) It is recommended to use two-step PCR program, the annealing temperature should be 58-64℃ as the reference range, and the annealing temperature can be increased in case of non-specific reaction. If you can't get good results due to the use of primers with low Tm value, you can try three-step PCR amplification, and the annealing temperature should be set in the range of 56℃-64℃ as a reference. The annealing and extension times for several common instruments are shown in the following table: 20 s for Roche, BioRad, Agilent, Hongshi, Dongshenglong, etc. 30 s for ABI 7000/7300/7500. The annealing/extension times can be set according to the different types of instruments and templates, please follow the instructions of the instruments. The annealing/extension time can be set according to different models of instruments and templates, please follow the instruction manual of the instrument... Read More | Inquire | 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 | Biochemical Test:SDS-PAGE (purity > 80%); Western blot with patient sample.Calculated Isoelectric Point:pH 8.38 | Product introduction：The additive contains a variety of cytokines, human albumin and other components. In order to ensure the activity, it is recommended that the additive part be thawed once and then sub packaged and frozen again. It is not advisable to freeze and thaw repeatedly. The Product introduction：The additive contains a variety of cytokines, human albumin and other components. In order to ensure the activity, it is recommended that the additive part be thawed once and then sub packaged and frozen again. It is not advisable to freeze and thaw repeatedly. The product contains no animal serum or animal serum components, no animal protein components, and no antibiotics.Matters needing attention：1. try to reduce the number of repeated freezing and thawing to avoid efficiency decline. 2. it is not suitable to place it at room temperature for a long time. 3. pay attention to aseptic operation and try to avoid pollution. 4. please wear experimental clothes and disposable gloves for operationScope of application：Cell culture additives... Read More |