| 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 | 2x Taq MasterMix is a premixed system composed of Taq DNA Polymerase, Mg2+, dNTPs, PCR stabilizers, and enhancers. The pre prepared PCR mixture makes the operation simpler and faster, and can minimize human error and contamination to the greatest extent possible. The original MasterMix formula 2x Taq MasterMix is a premixed system composed of Taq DNA Polymerase, Mg2+, dNTPs, PCR stabilizers, and enhancers. The pre prepared PCR mixture makes the operation simpler and faster, and can minimize human error and contamination to the greatest extent possible. The original MasterMix formula results in high yield, strong repeatability, and good stability of amplified products. This product does not contain dyes. After the PCR program is completed, an appropriate amount of sample buffer can be added as needed for electrophoresis operation. The amplified PCR product has an "A" base attached to the 3 'end, making it suitable for direct use in T/A cloning. Mainly suitable for PCR amplification of DNA, DNA sequencing and other experiments.Quality control: T665627Component5mlStorageT665627A2×Taq MasterMix5×1ml-20℃. Avoid freeze/thaw cycle.T665627BddH₂O5×1ml-20℃. Avoid freeze/thaw cycle.Notes: 2×Taq MasterMix contains Taq DNA Polymerase, 3mM MgCl2 and 400µM each dNTP After testing, there was no exogenous nuclease activity; PCR method for detecting residual DNA without host; Can effectively amplify single copy genes from multiple genomes.Usage:The following is an example of a PCR reaction system and reaction conditions for amplifying a 1 kb fragment using human genomic DNA as a template. In practical operation, corresponding improvements and optimizations should be made based on the template, primer structure, and target fragment size.1. PCR reaction systemReagent50 µlReaction systemFinal concentration2×Taq MasterMix25 µl1×Forward Primer，10 µM2 µl0.4 µMReverse Primer，10 µM2 µl0.4 µMTemplate DNA<0.5 µg<0.5 µg/50 µlddH2Oup to 50 µl/Attention: The primer concentration should be between 0.1 and 1.0 as the final concentration µ M serves as a reference for setting the range. In the case of low amplification efficiency, the concentration of primers can be increased; When non-specific reactions occur, the primer concentration can be reduced to optimize the reaction system.2. PCR reaction conditionsStepTemperatureTime/Pre denaturation95℃2 min/Denaturation94℃30 s25-35 cyclesAnneal55-65℃30 s25-35 cyclesExtend72℃30 s25-35 cyclesFinally extended72℃2 min/Attention:1) In general experiments, if the annealing temperature is 5 ℃ lower than the melting temperature Tm of the amplification primer, and the ideal amplification efficiency cannot be achieved, the annealing temperature should be appropriately reduced; When non-specific reactions occur, increase the annealing temperature to optimize the reaction conditions.2) The extension time should be set according to the size of the amplified fragment. The amplification efficiency of Taq DNA Polymerase in this product is 2 kb/min.3) The number of cycles can be set based on the downstream application of the amplification product. If the number of cycles is too small, the amplification amount is insufficient; If there are too many cycles, the probability of mismatches will increase, and non-specific backgrounds will be severe. So, while ensuring product yield, the number of cycles should be minimized as much as possible... Read More | Product Application：Isoelectric point: 7.2 (Maehly 1955).Inhibitors: Horseradish peroxidase is reversibly inhibited by cyanide and sulfide at a concentration of 10-5 M (Theorell 1951).Specificity: The enzyme exhibits a high specificity. Activity is observed with H2O2, MeOOH, and EtOOH (MaehlyProduct Application：Isoelectric point: 7.2 (Maehly 1955).Inhibitors: Horseradish peroxidase is reversibly inhibited by cyanide and sulfide at a concentration of 10-5 M (Theorell 1951).Specificity: The enzyme exhibits a high specificity. Activity is observed with H2O2, MeOOH, and EtOOH (Maehly and Chance 1954). See also Chmielnicka et al. (1971) and Morrison and Bayse (1973)... Read More | Carboxypeptidase B catalyzes hydrolysis of the basic amino acids lysine, arginine and histidine from theC-terminal end of polypeptides. The molecular weight is 34,500 daltons, the pH optimum is 8.0, and pI is 6.0.Carboxypeptidase B is competitively inhibited by arginine and lysine. The enzyme is Carboxypeptidase B catalyzes hydrolysis of the basic amino acids lysine, arginine and histidine from theC-terminal end of polypeptides. The molecular weight is 34,500 daltons, the pH optimum is 8.0, and pI is 6.0.Carboxypeptidase B is competitively inhibited by arginine and lysine. The enzyme is also inhibited by metal chelating agents, e.g., EDTA. Recombinant Carboxypeptidase B (EC 3.4.17.2) is expressed in E.Coli and purified by high pressure liquid chromatography. There is no trace of other enzyme (such as carboxypeptidase A and chymotrypsin) activity. No protease inhibitors such as PMSF are present in the preparation.Animal origin free:eliminate the risk of virus presence, or of any other potential adventitious agents found in animal-derived carboxypeptitase B.Stability:A sterile recombinant carboxypeptidase B lyophilized eliminates the risk of contamination and decreases the chances of activity loss in the process of transport and storage. High purity:1) Recombinant carboxypeptidase B provides increased specific activity and eliminates contaminating protease activities found in extracted enzymes with lower purity level. 2) No other contaminating proteases such as chymotrypsin and carboxypeptidase A. 3）Less than 10ppm of recombinant trypsin... Read More |