| Description | F665646 Component 5 ml 40 ml Storage F665646A 2×Flash Hot Start MasterMix (Dye) 5×1 mL 40×1 mL -20℃. Avoid freeze/thaw cycle. F665646B ddH₂O 5×1 mL 40×1 mL -20℃. Avoid freeze/thaw cycle.Products IntroductionThis product is a premixed system consisting of a F665646 Component 5 ml 40 ml Storage F665646A 2×Flash Hot Start MasterMix (Dye) 5×1 mL 40×1 mL -20℃. Avoid freeze/thaw cycle. F665646B ddH₂O 5×1 mL 40×1 mL -20℃. Avoid freeze/thaw cycle.Products IntroductionThis product is a premixed system consisting of a new type of highly efficient DNA polymerase, PCR Buffer, Mg2+, dNTPs, and PCR stabilizers and enhancers at a concentration of 2 ×. It contains a new type of high efficient hot starter enzyme, which can effectively inhibit the non-specific annealing of primers and non-specific amplification caused by primer dimer under low temperature. The product has a very high amplification speed and stability, the extension speed can be up to 5-15 sec/kb, suitable for rapid PCR reaction, the original MasterMix formula makes the whole reaction system very stable, more than 98% of the PCR amplification can be successful at once, at the same time, complex templates can also be amplified effectively, and can minimize the human error and contamination. The product has added dye (blue), and can be directly detected by electrophoresis after the reaction. Most of the amplified PCR products have an "A" base at the 3′ end, so they can be used directly for T/A cloning. It is mainly used for rapid PCR reactions and gene cloning where high fidelity is required.Quality controlTested to be free of exogenous nuclease activity; PCR method detects no host residual DNA; can efficiently amplify a wide range of baseSingle-copy genes in the genome.UsageThe following is an example of a PCR reaction system and reaction conditions using human genomic DNA as a template, and the actual operation should be based on the template.Plates, primer structures and target fragment sizes were improved and optimized accordingly.PCR reaction systemNote: Please use the final concentration of 0.1-1.0 µM as a reference for setting the range of primer concentration. If the amplification efficiency is not high, the primer can be increased.concentration; when a non-specific reaction occurs, the primer concentration can be reduced, thus optimizing the reaction system.PCR reaction conditionsNote: If the amplified sample is bacterial liquid, add "pre-denaturation 95℃ for 5min" step.Optimization of parameter settings1. Template DNA amount setting:Excessive amounts of template may result in non-specific amplification or smear. The recommended amount of template DNA in a 50 µl PCR reaction system is as follows:-Human genomic DNA 5 ng-500 ng-Escherichia coli genomic DNA 50 pg-100 ng-plasmid DNA 10 pg-1 ng2. Primer concentration setting:The primer concentration can be set between 0.1 µM and 1.0 µM. Too low a primer concentration may result in low amplification products. Too high a primer concentration will inhibit specific amplification and may result in non-specific amplification.3. Annealing temperature setting: In general, the annealing temperature in the experiment is 5℃ lower than the melting temperature of the amplification primer, Tm, which is not able to get the ideal amplification efficiency.The annealing temperature can be lowered appropriately in the case of non-specific reactions; the annealing temperature can be increased appropriately in the case of non-specific reactions. For complex templates, the annealing temperature needs to be adjusted to achieve efficient amplification.4. Extension time setting: The extension time should be set according to the size of the amplified fragments. The following are recommended extension times:Simple templates such as plasmids: 5-15 s/kb; Conventional genome, cDNA templates: 10-15 s/kb; Complex templates, crude extraction templates: 20-30 s/kb;(The extension time should not be too short it should be at least 5 s/kb and not more than 30 s/kb).5. Cycle number setting: The number of cycles can be set according to the downstream application of the amplified product. If the number of cycles is too low, the amount of amplification will be insufficient; if the number of cycles is too high, the chance of mismatch will increase and the non-specific background will be serious. Therefore, the number of cycles should be minimized under the premise of ensuring the product yield... Read More | Inquire | Amine-Reactive probe which passively diffuse into cells and it is nonfluorescent until the acetate groups are cleaved by intracellular esterases to yield the highly fluorescent, amine-reactive fluorophore. Upon reaction with amine-containing residues of intracellular proteins, these probes form dye Amine-Reactive probe which passively diffuse into cells and it is nonfluorescent until the acetate groups are cleaved by intracellular esterases to yield the highly fluorescent, amine-reactive fluorophore. Upon reaction with amine-containing residues of intracellular proteins, these probes form dye protein adducts that are well retained in cells as they move and divide during embryonic development.A Non-fluorescent cell permeant amine-reactive probe for long term tracing of cell... Read More | Product contentF665774Component5 mLStorageF665774A2×Fast Probe Mixture5×1 mL-20℃. Avoid freeze/thaw cycle.F665774B50×High ROX200 µL-20℃. Avoid freeze/thaw cycle.F665774CddH2O5×1 mL-20℃. Avoid freeze/thaw cycle.Product IntroductionFast Probe Mixture is a preProduct contentF665774Component5 mLStorageF665774A2×Fast Probe Mixture5×1 mL-20℃. Avoid freeze/thaw cycle.F665774B50×High ROX200 µL-20℃. Avoid freeze/thaw cycle.F665774CddH2O5×1 mL-20℃. Avoid freeze/thaw cycle.Product IntroductionFast Probe Mixture is a pre-mixed system for real-time fluorescence PCR by probe method (TaqMan, Molecular Beacon, etc.), with a concentration of 2×, including Fast Taq DNA Polymerase, PCR Buffer, dNTPs, Mg2+ and so on, which is easy and convenient to operate. It is mainly used for the detection of genomic DNA target sequence and cDNA target sequence after RNA reverse transcription. The Fast Taq DNA Polymerase contained in this product can effectively reduce the non-specific amplification generated by the non-specific binding of primers and templates or primer dimerization at room temperature, and the activation of the enzyme only needs to be incubated at 95 ℃ for 30 s. The whole PCR reaction process can save about 40 minutes compared with the ordinary reaction, which greatly shortens the reaction time of PCR. The combination of unique PCR buffer system and fast hot start enzyme effectively inhibits the generation of non-specific products and significantly improves the PCR amplification efficiency with stronger fluorescence signal, higher sensitivity and wider linear range. The product has a wide range of applications and can be used for both normal and rapid quantitative PCR programs.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 (F665766):Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96 and others.Instruments that require Low ROX calibration (F665768):ABI Prism7500/7500 Fast, QuantStudio®3 System, QuantStudio®5 System, QuantStudio®6 Flex System, QuantStudio®7 Flex System, ViiA 7 system. Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000, and more.Instruments that require High ROX calibration (F665774):ABI Prism 7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, and others.matters needing attention1. Before use, please mix gently by turning up and down, avoid foaming as much as possible, and use after brief centrifugation.2. Avoid repeated freezing and thawing of this product, repeated freezing and thawing may degrade the product performance. This product can be stored for long term at -20℃, protected from light. If frequent use is required within a short period of time, it can be stored at 2-8℃.UsageThe following examples are conventional PCR reaction systems and reaction conditions, which should be improved and optimized according to the template, primer structure and target fragment size in actual operation.1.PCR reaction systemreagents50µl reaction systemfinal concentration2×Fast Probe Mixture25 µl1×Forward Primer, 10µM1µl0.2µM¹⁾Reverse Primer, 10µM1µl0.2µM¹⁾Probe, 10 µM1µl0.2µM²⁾Template DNA2µl³⁾ 50x Low ROX or High ROX(optional)⁴⁾1µl1×ddH₂Oup to 50µlNote: 1) Usually the primer concentration of 0.2µM can get better results, and 0.1-1.0µM can be used as a reference for setting the range. 2) The final concentration of the probe used is related to the fluorescent quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance, so please refer to the instruction manual of the instrument or the specific requirements of the use of each fluorescent probe for the adjustment of the concentration in actual use.(3) Usually the amount of DNA template is 10-100ng genomic DNA or 1-10ng cDNA as a reference. Since the templates of different species contain different copy numbers of target genes, the templates can be subjected to gradient dilution to determine the optimal amount of template to be used.(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.2. PCR reaction program:A two-step PCR reaction program is recommended, and this program is set up using the ABI 7500 Fluorescent Quantitative PCR Instrument as a reference.Note: 1) The enzyme used in this product must be pre-denatured at 95°C for 30s to achieve enzyme activation. 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 1-4 minutes to allow the starting template to fully unchain.(2) It is recommended to use two-step PCR reaction program, if you do not get good experimental results due to the use of primers with lower Tm values, etc., you can try to carry out three-step PCR amplification, and the annealing temperature, please use the range of 56 ℃ - 64 ℃ as a setting reference... Read More | Purity>97% by SDS-PAGE and HPLC analyses.Additional sequence informationFunction N-terminal glycine. Full-length mature chain lacking the signal peptideFunctionHas chemotactic activity for neutrophils. May play a role in inflammation and exerts its effects on endothelial cells in an autocrine Purity>97% by SDS-PAGE and HPLC analyses.Additional sequence informationFunction N-terminal glycine. Full-length mature chain lacking the signal peptideFunctionHas chemotactic activity for neutrophils. May play a role in inflammation and exerts its effects on endothelial cells in an autocrine fashion. In vitro, the processed forms GRO-alpha(4-73), GRO-alpha(5-73) and GRO-alpha(6-73) show a 30-fold higher chemotactic activity.Post-translationalN-terminal processed forms GRO-alpha(4-73), GRO-alpha(5-73) and GRO-alpha(6-73) are produced by proteolytic cleavage after secretion from peripheral blood monocytes... Read More |