| Description | Protease S. aureus, V8 (endoproteinase-Glu-C) specifically cleaves peptide bonds on the COOH-terminal side of either aspartic or glutamic acids (Drapeau et al. 1972). Houmard and Drapeau (1972) report that in the presence of ammonium buffers the enzyme specificity can be limited to glutamoyl bonds. Protease S. aureus, V8 (endoproteinase-Glu-C) specifically cleaves peptide bonds on the COOH-terminal side of either aspartic or glutamic acids (Drapeau et al. 1972). Houmard and Drapeau (1972) report that in the presence of ammonium buffers the enzyme specificity can be limited to glutamoyl bonds. Its rather unique specificity, which can be considered as opposite to that of trypsin, makes it a useful tool for protein chemistry and peptide mapping studies (Cleveland et al. 1977) and (Hall et al. 1978).Characteristics of Protease from S. aureus, V8:Characteristics of Protease, Staph aureus (Endoproteinase Glu-C)Molecular Weight27,000 (Drapeau 1978).Optimal pH4.0 and 7.8 with hemoglobin substrate. (Drapeau et al. 1972).Extinction Coefficientextinction coefficient = 4.26 (Houmard 1976).InhibitorsDiisopropyl fluorophosphate (DFP) and monovalent anions such as F-, Cl-, Br-, CH3COO-, and NO3- (Houmard 1976).MethodEnzyme activity is determined by the casein digestion assay described by Drapeau (1976). Since substrate grade caseins can vary from lot to lot and according to the manner produced, the standardization of casein digestion assays has been difficult to achieve. The use of a reference enzyme preparation is suggested. One unit is that amount of enzyme which releases acid soluble fragments equivalent to 0.001 A280 per minute at 37°C and pH 7.8 under the specified conditions.Reagents1% Casein in 0.05 M Tris⋅PO4 buffer, pH 7.8. (Dissolve 1 gram "Hammersten" casein in 50 ml 0.01 N NaOH with gentle heating and stirring. Add 40 ml reagent grade water and 5.0 ml 1.0 M Tris. Adjust pH to 7.8 with H3PO4 and q.s. to 100 ml.)10% Trichloroacetic acid (TCA)EnzymeDissolve at 1 mg/ml in reagent grade water.ProcedureEquilibrate a series of tubes with 5.0 ml of 1% casein at 37°C for 5 minutes. At zero time add 10 ul or 20 ul of enzyme. Mix. Include a reagent blank. Exactly ten minutes after adding sample, stop reaction by adding 5.0 ml TCA. Mix. Allow tubes to stand ten minutes and then filter. Read A280 of the filtrate.Calculation... Read More | Taq MasterMix is a premixed system composed of Taq DNA Polymerase, Mg2+, dNTPs, as well as PCR stabilizers and enhancers, with a concentration of 2 ×. The pre prepared PCR mixture makes the operation simpler and faster, and can minimize human error and contamination to the greatest extent Taq MasterMix is a premixed system composed of Taq DNA Polymerase, Mg2+, dNTPs, as well as PCR stabilizers and enhancers, with a concentration of 2 ×. 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 has been added with a dye (blue), and can be directly subjected to electrophoresis detection after the reaction is completed. The amplified PCR product has an "A" base attached to its 3 'end, making it suitable for direct use in T/A cloning. Mainly suitable for PCR amplification of DNA, DNA sequencing and other experiments. T665590Component5 mL25 mLStorageT665590A2×Taq MasterMix (Dye)5×1 mL5×5 mL-20℃. Avoid freeze/thaw cycle.T665590BddH₂O5×1 mL5×5 mL-20℃. Avoid freeze/thaw cycle.2×Taq MasterMix contains Taq DNA Polymerase, 3 mM Mg Cl₂ 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.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×Taq 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 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 ° C 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 | Inquire | Inquire | Inquire |