| Description | UltraBio™ Multiple Amplification Mix is a universal multiplex amplification reagent that can be used for constructing multiplex amplicon libraries for microbial/genetic mutation detection. It is compatible with ultra-multiplex amplification, with a primer number range of 1 to 1500, amplicon UltraBio™ Multiple Amplification Mix is a universal multiplex amplification reagent that can be used for constructing multiplex amplicon libraries for microbial/genetic mutation detection. It is compatible with ultra-multiplex amplification, with a primer number range of 1 to 1500, amplicon length range of 100 to 1800 bp, GC content range of 13% to 75%, and a minimum template input amount of 1 ng. The reagent undergoes strict background microbe quality control to minimize false positives from non-specific amplification. Rigorous functional validation ensures high stability and reproducibility, making it ideal for NGS-based multiplex targeted amplification in microbial/genetic mutation detection.Self-Supplied Materials1. Instruments PCR thermal cycler, Pipettes, Magnetic stand, Microcentrifuge, Vortex mixer, Agarose gel electrophoresis system, UV gel imaging system, Agilent Bioanalyzer 2100 (or equivalent).2. Reagents Agarose, 1× TAE buffer, Nucleic acid dye, DNA marker, Ampure XP beads (or equivalent), Freshly prepared 80% ethanol, Nuclease-free water.3. Consumables 1.5 mL microcentrifuge tubes, 0.2 mL PCR tubes, DNase/RNase-free pipette tips.ProtocolI. Multiplex Amplification & Product VerificationReagent PreparationThaw UltraBio™ Multiplex Amplification Mix on ice.Vortex briefly and centrifuge before use.Reaction Setup (10 µL Standard Volume)ComponentVolume per ReactionFinal ConcentrationUltraBio™ Multiplex Amplification mix2.5 µL4xPrimer Mix1 µL20nM (2.5~100nM)Template DNAX µL10ng (1~100ng)Nuclease-free waterUp to10 µL/Total10 µL/Note:Total reaction volume can be scaled to 10–50 µL (adjust mix proportionally).Primer concentration and annealing temperature (T) should be optimized for each panel.Mixing & CentrifugationVortex gently and centrifuge briefly to collect liquid.PCR Cycling Program*T: Annealing temperature depends on primer panel (optimize empirically).Cycle number: Adjust based on template input (default: 25 cycles).Product QC: Analyze amplification success via 2% agarose gel electrophoresis or Agilent 2100 Bioanalyzer.II. Amplicon Purification (Ampure XP Beads)Bead EquilibrationWarm Ampure XP beads to room temperature; vortex thoroughly.Bead BindingDilute PCR products to 50 µL with nuclease-free water.Add 60 µL beads (1.2× ratio), vortex, and incubate 5 min at RT.Wash StepsPlace tubes on a magnetic stand for 3–5 min; discard supernatant.Wash twice with 200 µL fresh 80% ethanol (30 sec per wash).Air-dry beads for 2–3 min until matte.ElutionResuspend beads in 22 µL nuclease-free water; incubate 2 min at RT.Transfer 20 µL supernatant to a new PCR tube. Store purified products at –20°C... 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 | Purity> 95 % by SDS-PAGE and HPLC analyses.FunctionPromotes cell proliferation, chemotaxis, angiogenesis and cell adhesion. Appears to play a role in wound healing by up-regulating, in skin fibroblasts, the expression of a number of genes involved in angiogenesis, inflammation and matrix Purity> 95 % by SDS-PAGE and HPLC analyses.FunctionPromotes cell proliferation, chemotaxis, angiogenesis and cell adhesion. Appears to play a role in wound healing by up-regulating, in skin fibroblasts, the expression of a number of genes involved in angiogenesis, inflammation and matrix remodeling including VEGA-A, VEGA-C, MMP1, MMP3, TIMP1, uPA, PAI-1 and integrins alpha-3 and alpha-5. CYR61-mediated gene regulation is dependent on heparin-binding. Down-regulates the expression of alpha-1 and alpha-2 subunits of collagen type-1. Promotes cell adhesion and adhesive signaling through integrin alpha-6/beta-1, cell migration through integrin alpha-v/beta-5 and cell proliferation through integrin alpha-v/beta-3.Banckground:Cyr61, also known as CCN1, is a 40-45 kDa matricellular glycoprotein that plays an important role in cellular adhesion and migration (1). Cyr61 consists of an IGFBP domain, a VWF type C domain, a TSP type I domain, and a cysteine knot domain (2). Mature human Cyr61 shares 93% amino acid sequence identity with mouse and rat Cyr61. It is widely expressed during development and in adult tissues (2, 3). Cyr61 associates with the extracellular matrix (ECM) and with many cell surface molecules including Integrins alpha V beta 3, alpha V beta 5, alpha M beta 2, and alpha 6 beta 1, Syndecan-4, and heparan sulfate proteoglycans (1, 3). Cyr61 mediates the adhesion and migration of multiple cell types and also promotes vascular endothelial cell tubule formation (4-6). Plasmin cleavage of ECM-bound Cyr61 releases a 28 kDa N-terminal fragment which retains the ability to promote endothelial cell migration (7). Cyr61 exhibits both tumorigenic and tumor suppressor properties. It is up-regulated and promotes tumorigenesis, angiogenesis, and metastasis in breast, renal, gastric, squamous cell, and colorectal carcinomas as well as in glioma (8-12). In contrast, whendown-regulated, it suppresses tumor growth in endometrial, hepatic, and non-small cell lung cancers (8, 13, 14). Cyr61 is also up-regulated in injured skin and bone where it induces the expression of growth factors, cytokines, proteases, and integrins involved in wound repair (15, 16)... Read More | Purity>95% (SDS-PAGE&HPLC) Endotoxin level<0.1 EU/µgFunctionMay regulate apoptosis, cell proliferation and cell differentiation. Binds beta-galactoside and a wide array of complex carbohydrates. Inhibits CD45 protein phosphatase activity and therefore the dephosphorylation of Lyn Purity>95% (SDS-PAGE&HPLC) Endotoxin level<0.1 EU/µgFunctionMay regulate apoptosis, cell proliferation and cell differentiation. Binds beta-galactoside and a wide array of complex carbohydrates. Inhibits CD45 protein phosphatase activity and therefore the dephosphorylation of Lyn kinase.Gal-1 is also engaged in many protein-protein interactions. Gal-1 plays a number of crucial roles in neuronal cell differentiation and survival in both the central and the peripheral nervous systems, and the establishment and maintenance of T-cell tolerance and homeostasis in vivo... Read More |