| Description | ApplicationIt is used for the development and mass preparation of total bile acid (TBA) reagents.Enzymatic properties (note that 2+, +, 3+ are superscripts)Source: MicroorganismEnzymology Committee Number: EC1.1.1.50Molecular weight: 30 kDa (SDS-PAGE)Isoelectric point: 5.2Km value: 3.6×10-5M (ApplicationIt is used for the development and mass preparation of total bile acid (TBA) reagents.Enzymatic properties (note that 2+, +, 3+ are superscripts)Source: MicroorganismEnzymology Committee Number: EC1.1.1.50Molecular weight: 30 kDa (SDS-PAGE)Isoelectric point: 5.2Km value: 3.6×10-5M (androsterone), 4.7×10-5M (NAD+)Inhibitors: Cu²⁺，Ag⁺，Hg²⁺，Zn²⁺，Fe³⁺ Optimum pH: 8.5-9.5 Figure 1Optimum temperature: 55℃ Figure 2pH stability: 6.0-9.0 (25℃,16h) Figure 3Thermal stability: Stable below 37℃ (pH9.0, 30min) Figure 4Stability: -25 ~ -15℃ standing store for 12 monthsMore than 90% activity Figure 5Protective agent: BSA Assay method for activity1. PrincipleThe NADH produced by the reaction can be detected with a spectrophotometer at 340nm.2. Definition of enzyme activityUnit enzyme activity is defined as the amount of enzyme required to catalyze the production of 1µmolNADH per minute under the following conditions.3. Reagent preparationReagent I: 0.1M sodium pyrophosphate (adjust pH to 8.9 with HCl).Reagent II: Dissolve 319mg NAD+ into 25mL double steaming water, adjust the pH to 7.0-7.5 with solid NaHCO3, and adjust the volume to 30mL with double steaming water.Reagent III: Dissolve 30mg into 100mL methanol.Enzyme diluent: 10mMTris-HCl, pH 9.0.4. Operation procedure1. Add 2.6mL reagent I, 0.2mL reagent II and 0.1mL reagent III into a 3mL colorimetric dish and mix well.2. Preheat the reaction mixture at 25°C for 5min.3. Add 0.1mL enzyme liquid to the reaction mixture, mix it well, react at 25°C, and record the absorbance change within 1min at 340nm with a spectrophotometer (∆As).* Replace enzyme liquid with enzyme diluent, other steps are the same, the absorbance of the resulting solution is blank absorbance (∆Ab)∆A=∆As-∆Ab5. Vitality computing3.00: total volume of reaction liquid (mL);0.10: enzyme liquid volume (mL);1.0: optical path length (cm);df: dilution ratio;C: Enzyme concentration (mg/mL);6.22: Nanomolar absorption coefficient of NADH at 340nm (cm2/µmol)... Read More | Inquire | Inquire | Es Taq DNA Polymerase is an optimized mixed enzyme of Taq and Pfu DNA Polymerase, with 5 '→ 3' DNA polymerase activity, 5 '→ 3' exonuclease activity, and 3 '→ 5' exonuclease activity. Compared with Taq DNA Polymerase, Es Taq DNA Polymerase has excellent performance of high Es Taq DNA Polymerase is an optimized mixed enzyme of Taq and Pfu DNA Polymerase, with 5 '→ 3' DNA polymerase activity, 5 '→ 3' exonuclease activity, and 3 '→ 5' exonuclease activity. Compared with Taq DNA Polymerase, Es Taq DNA Polymerase has excellent performance of high amplification efficiency and low mismatch rate, and can efficiently amplify DNA fragments. Most of the PCR products amplified with this product contain an "A" base at the 3 'end, which can be directly used for T/A cloning. This product is suitable for conventional PCR reactions and gene cloning reactions that require high fidelity. E665597Component500 UStorageE665597AEs Taq DNA Polymerase, 5 U/µL 100 µL -20℃. Avoid freeze/thaw cycle.E665597B10×PCR Buffer 1.8 mL -20℃. Avoid freeze/thaw cycle.Activity definition:Using activated salmon sperm DNA as a template/primer, the amount of enzyme required to incorporate 10 nmol of deoxyribonucleotide into acidic insoluble substances is defined as 1 active unit (U) at 74 ℃ for 30 minutes.Quality control:After multiple column purifications, SDS-PAGE detected a purity of over 99%; No exogenous nuclease activity detected; PCR method for detecting residual DNA without host; Can effectively amplify single copy genes in the human genome; Store at room temperature for one month without significant changes in activity.1. PCR reaction system Reagent 50 µlReaction system Final concentration 10×PCR Buffer 5 µL 1× dNTP Mix，10 mM each 1 µL 200 µM each 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 Es Taq DNA Polymerase，5 U/µl 0.25-0.5 µl 1.25-2.5U/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 94℃ 2 min / Denaturation 94℃ 30 s 25-35 cycles Anneal 55-65℃ 30 s 25-35 cycles Extend 72℃ 30 s 25-35 cycles Finally extended 72℃ 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 Es 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 | Inquire |