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 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