PCR premix liquid of fluorescent quantitative PCR thermal cycler and preparation method and application of PCR premix liquid
A fluorescence quantitative, thermal cycler technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., to avoid false positive results, easy identification, and reduce pre-reaction time.
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[0036] The preparation method of described premix is as follows:
[0037] 1) Prepare KCl solution, Tris solution, (NH 4 ) 2 SO 4 solution, MgCl 2 Dissolve, vortex and mix well, store at 4°C for later use;
[0038] 2) Then sterile double distilled water, and the KCl solution prepared in step 1), Tris solution, (NH 4 ) 2 SO 4 solution, and MgCl 2 The solution is pre-mixed in a sterilized storage tube, the pH of the solution is adjusted to 8.5, sterilized after mixing to obtain a pretreatment mixture; the KCl content is 40 μmol to 80 μmol; the (NH 4 ) 2 SO 4 The content is 0-4 μmol; the content of the Tris solution is 10 μmol to 30 μmol; the MgCl 2 The content is 2μmol~4μmol;
[0039] 3) Add 1.8μmol~3μmol dNTPs, 40U~100U DNA polymerase, 5μmol~20μmol dye SYTO13, 30pmol~120pmol reference fluorescent CXR, 0-100μL DMSO and 0-80μL propane to the pretreatment mixture obtained in step 2). Triol, mix well and centrifuge, finally add sterile double distilled water to 1mL, mi...
Embodiment 1
[0042] Add 1 μL of DNA intercalating fluorescent dye SYTO13 (5 mM), 1 μL of reference fluorescent dye CXR (30 μmol), 80 μL of KCl solution (1mol / L), 80 μL of glycerol, 60 μL of DMSO, 10 μL of Tris solution (1mol / L), MgCl 2 Solution (0.1mol / L) 20μL, dNTPs solution 45μL, hot start DNA polymerase (2.5U / μL) added 16μL
[0043] Preparation process:
[0044] Sterilization of equipment and consumables - inorganic compounds KCl, Tris, MgCl 2 Preparation of solution—sterilization of inorganic compound solution (temperature 126°C, time 15min)—cooling storage (4°C)—ingredient 1 (add 200 μL double distilled water, KCl solution, Tris solution, MgCl 2 solution)—high concentration of HCl to adjust the pH of the solution to 8.5——2500r / min vortex for 30s, 4000×g centrifugation for 30s——inorganic compound solution sterilization (temperature 126°C, sterilization 15min)——cooling storage (4°C) ——Ingredient 2 (add dNTPs, DNA polymerase, dye SYTO13, glycerol, DMSO in sequence)——2500r / min vortex fo...
Embodiment 2
[0046] Add 2.4 μL of DNA intercalating fluorescent dye SYTO13 (5 mM), 2 μL of reference fluorescent dye CXR (30 μmol), 80 μL of KCl solution (1mol / L), 80 μL of glycerol, 100 μL of DMSO, 25 μL of Tris solution (1mol / L), MgCl 2 (0.1mol / L) solution 30μL, dNTPs solution 60μL, hot start DNA polymerase (2.5U / μL) added 20μL
[0047] Preparation process:
[0048] Sterilization of equipment and consumables - inorganic compounds KCl, Tris, MgCl 2 Preparation of solution—sterilization of inorganic compound solution (temperature 126°C, time 15min)—cooling storage (4°C)—ingredient 1 (add 200 μL double distilled water, KCl solution, Tris solution, MgCl 2 solution)—high concentration of HCl to adjust the pH of the solution to 8.5——2500r / min vortex for 30s, 4000×g centrifugation for 30s——inorganic compound solution sterilization (temperature 126°C, sterilization 15min)——cooling storage (4°C) ——Ingredient 2 (add dNTPs, DNA polymerase, dye SYTO13, reference fluorescent CXR, glycerol, DMSO in ...
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