[Objective] The experiment aimed to study an efficient method of Nuclei extraction of cotton and provided technical support for constructing large-insert genomic library and sequencing complete genome. [Method] The co...[Objective] The experiment aimed to study an efficient method of Nuclei extraction of cotton and provided technical support for constructing large-insert genomic library and sequencing complete genome. [Method] The cotton cotyledons germinated in dark moisture chamber for one week were chopped with a sharp sterile scalpel in a Petri dish which contained ice-cold nucleus isolation buffer (10 mmol/L MgSO4, 5 mmol/L KCl, 0.5 mmol/L HEPES, 1 mg/ml DTT, 0.25% Triton X-100 and 2% PVP40), then the nuclei were collected after selected through 100, 50 and 30 μm nylon meshes and centrifugation. Meanwhile, the tender leaves and cotyledons with different germination time in dark were treated by grinding method and sharp scalpel method. [Result] The chopping with a sharp scalpel method was very simple and rapid, which did not need grind and mercaptoethanol treatment and the successful extraction rate was 100%.[Conclusion] An efficient method of nuclei extraction of cotton with simple, high efficiency, rapid reaction and poison free were established.展开更多
基金Supported by the National Natural Science Foundation of China(No.30170501)the State of New Varieties of GMO Cultivation Major Projects (No.2008ZX08005-003)the National High-tech Research Development Plan (No.2003AA207051)~~
文摘[Objective] The experiment aimed to study an efficient method of Nuclei extraction of cotton and provided technical support for constructing large-insert genomic library and sequencing complete genome. [Method] The cotton cotyledons germinated in dark moisture chamber for one week were chopped with a sharp sterile scalpel in a Petri dish which contained ice-cold nucleus isolation buffer (10 mmol/L MgSO4, 5 mmol/L KCl, 0.5 mmol/L HEPES, 1 mg/ml DTT, 0.25% Triton X-100 and 2% PVP40), then the nuclei were collected after selected through 100, 50 and 30 μm nylon meshes and centrifugation. Meanwhile, the tender leaves and cotyledons with different germination time in dark were treated by grinding method and sharp scalpel method. [Result] The chopping with a sharp scalpel method was very simple and rapid, which did not need grind and mercaptoethanol treatment and the successful extraction rate was 100%.[Conclusion] An efficient method of nuclei extraction of cotton with simple, high efficiency, rapid reaction and poison free were established.
