| Description | This product is a premixed system composed of GoldStar Best DNA Polymerase, Mg2+, dNTPs, PCR stabilizers and enhancers, with a concentration of 2 ×. It has the advantages of simple and fast operation, high sensitivity, strong specificity, and good stability, which can minimize human error and This product is a premixed system composed of GoldStar Best DNA Polymerase, Mg2+, dNTPs, PCR stabilizers and enhancers, with a concentration of 2 ×. It has the advantages of simple and fast operation, high sensitivity, strong specificity, and good stability, which can minimize human error and pollution. The GoldStar Best DNA Polymerase contained in this product is a chemically modified hot start high fidelity polymerase. This polymerase has 5 '-3' DNA polymerase activity, 5 '-3' exonuclease activity, and 3 '-5' exonuclease activity. Under normal PCR conditions, compared with GoldStar Taq DNA polymerase, it has excellent performance of high amplification efficiency and low mismatch rate. The chemically modified enzyme does not exhibit polymerase activity at room temperature, effectively avoiding non-specific amplification caused by non-specific binding of primers and templates or primer dimers at room temperature. Enzyme activation requires incubation at 95 ℃ for 10 minutes, which can be integrated into existing PCR thermal cycling programs. The optimized buffer system maximizes the effectiveness of the enzyme, achieving high fidelity, specificity, amplification efficiency, and sensitivity for the target fragment. This product has been added with a dye (blue), and can be directly subjected to electrophoresis detection after the reaction is completed. Most PCR products obtained from amplification have an "A" base attached to the 3 'end, making them suitable for direct use in T/A cloning. Suitable for routine PCR reactions and gene cloning experiments that require high fidelity.G665556Component1 mL5 mLStorageG665556A2×GoldStar Best MasterMix (Dye)1 mL5×1 mL-20℃. Avoid freeze/thaw cycle.G665556BddH₂O1 mL5×1 mL-20℃. Avoid freeze/thaw cycle.Notes: 2×GoldStar Best MasterMix contains GoldStar Best DNA Polymerase, 3.4 mM MgCl₂ and 400 µM each dNTP.Quality control: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; Storage at 2-8 ℃ for three months showed no significant change in activity.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 system Reagent 50 µlReaction system Final concentration 2×GoldStar Best MasterMix（Dye） 25 µl 1× Forward Primer，10 µM 2 µl 0.4 µM Reverse Primer，10 µM 2 µl 0.4 µM Template DNA <0.5 µg <0.5 µg/50 µl ddH2O up 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 conditions Step Temperature Time / Pre denaturation 95℃ 10 min / Denaturation 94℃ 30 s 30-40 cycles Anneal 55-65℃ 30 s 30-40 cycles Extend 72℃ 60 s 30-40 cycles Finally extended 72℃ 5 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, it should be appropriately reduced Low annealing temperature; 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, and the GoldStar Best DNA Polymerase contained in this product should be The amplification efficiency is 1-2 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 the number of cycles is too highMany, 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.4) This product must achieve enzyme activation under pre denaturation conditions of 95 ℃ and 10 minutes... Read More | ProductsThis product is a high purity genomic DNA extract from 293T cells, agarose gel (0.7%) electrophoresis showed that the size of the DNA extract is more than 15Kb, and basically no degradation, the product is ultimately preserved in TE Buffer, which can be widely used in molecular biology ProductsThis product is a high purity genomic DNA extract from 293T cells, agarose gel (0.7%) electrophoresis showed that the size of the DNA extract is more than 15Kb, and basically no degradation, the product is ultimately preserved in TE Buffer, which can be widely used in molecular biology experiments, such as PCR, enzyme digestion, hybridization, microarray analysis, and other molecular biology experiments.The product was quantified using NanoDrop One at a concentration of 200 ng/µL.Preparation and precautions before useLong-term storage at -20˚C is recommended. Before use, the bottle should be removed from the refrigerator and equilibrated to room temperature and centrifuged before opening the cap for use. Samples should be restored to the sealed state as soon as possible after opening.How to use (take qPCR experiment as an example)1. Amplification template preparationThe samples to be detected were diluted with TE (10 mM Tris-Cl, pH 8.0,1 mM EDTA), and the concentration after dilution was as close as possible to the range of 0.05-10 ng/µL. The samples were placed on ice at 4°C and set aside.2. Standard dilution: according to the following table, firstly dilute Human DNA Standard 1 (100ng/uL) with TE to make 5 different concentrations of standards according to the table below. 10ng/µL of DNA Standard 1 (Std. 1) can be stored stably at -20℃ for 1 month; Std2-5 can only be used on the same day, and should be placed at 4℃ or on ice when not in use for the time being after preparation. When not used temporarily after preparation, it should be stored at 4℃ or on ice.styleCorresponding concentration (ng/µL)Minimum dilution volume (in µL)Std.11010 [100 ng/µL DNA Standard 1] + 90 TEStd.22.520 [Std. 1] +60 TEStd.30.62520 [Std. 2] +60 TEStd.40.1562520 [Std. 3] +60 TEStd.50.039062520 [Std. 4] +60 TE3. qPCR reaction system preparationThe cryopreserved reagents to be used were completely thawed and mixed by inversion several times before preparation, and then briefly centrifuged and prepared for use. 20 µL of the base reaction system was as follows.The base reaction system for 20 µL was as follows:reagents20µL reaction system2×qPCRMix10µLPrimerMixXµLProbeMixXµLTemplate4µLddH2OMake up to 20 µLNote: High Rox model: add 1 µL of 50×High Rox per 50 µL of reaction system; Low Rox model: add 1 µL of 50×High Rox per 500 µL of reaction system.Usually, better results can be obtained with a primer concentration of 0.2 µM, and 0.1-1.0 µM can be used as a reference for setting the range.The 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 manual of the instrument or the specific requirements for the use of each fluorescent probe for the adjustment of the concentration during actual use.Prepare a sufficient amount of reaction system mixture as required. After the reaction system has been prepared and mixed thoroughly, add 16 µL per well to the reaction wells. Then add the prepared standard and diluted sample into the corresponding reaction wells, the volume of addition is 4µL/well. TE was added to the blank control tube, and the same amount of TE was added at 4 µL/well.It is recommended to use 20 µL for the reaction, if you need to perform a smaller system reaction, reduce the system components in equal proportion.4. qPCR reaction programThe following is an example of our GoldStar Probe Mixture reaction conditions, which should be improved and optimized according to the PCR product template, primer structure and target fragment size.movetemptimingcirculatepremutability95°C10min1denaturation95°C10sec55Annealing/Extension60°C30sec5Data analysis1. Standard curve productionThe standard curve was plotted with reference to the Excel sheet for data processing. The correlation coefficient R2 of the standard curve should not be lower than 0.98, and the slope should be between -3.1 and -3.6 when the Ct value is the vertical coordinate. If the parameters of the standard curve are unreasonable, it is recommended to repeat the experiment... Read More | Inquire | Inquire | 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 |