Transgene mouse model of poly glumine disease, its culturing method and medicine for treating the disease
A technology of polyglutamine and transgenic mice, which is applied in the field of life sciences and can solve problems such as the unclear role of DRPLA
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[0126] 1. Molecular biology experiments
[0128] All the plasmids were amplified with DH5α as the host bacteria, and the method of plasmid extraction refers to molecular cloning. Restriction endonucleases from Biolab Company and T4 DNA ligase from Takara Company were used to digest and connect the plasmids according to the recommended procedures.
[0129] 2) PCR
[0130] 10x PCR Buffer: 166mM (NH 4 ) 2 SO 4 , 670mM Tris (pH 8.8), 67mM MgCl 2 , 50mM beta-mercaptoethanol, 67μM EDTA.
[0131] PCR Reaction: DNA template, Primer, 10xBuffer, DMSO(10x), dNTP, Taq, H 2 O.
[0132] Thermofile: 93°C 90 sec, (93°C 30sec, 57°C 30sec, 65°C 3min) x 40cycles, 65°C 10min, 4°C max.
[0133] 3) DNA extraction and Southern hybridization
[0134] Take 1-2cm of mouse tail and place it in 1.5ml lysis buffer (100mM NaCl, 100mM Tris (pH 8.0), 25mM EDTA, 0.5% (w / v) SDS), add 20μl proteinase K (10mg / ml), and lyse at 55℃ More than 8 hours, intermittent shaking. The ly...
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