RESUMO
A simple and rapid method for preparation of plasmid DNA from overnight incubation was introduced. It does not require any additional reagents; the incubation mixture containing recombinant plasmid DNA was just mixed with H2O and phenol/chloroform/isoamyl alcohol in certain ratio. After vortexing and spinning of the mixture, the supernatant could be directly loaded onto agarose gel and analyzed using electrophoresis. The whole preparation requires only 3-5 minutes. So to quickly screen recombinant clones, this method is better compared with traditional methods.
Assuntos
Centrifugação com Gradiente de Concentração , Clorofórmio , Química , Clonagem Molecular , Métodos , DNA Bacteriano , Genética , Metabolismo , Desoxirribonuclease HindIII , Metabolismo , Eletroforese , Escherichia coli , Genética , Pentanóis , Química , Fenol , Química , Plasmídeos , Química , Genética , Reprodutibilidade dos Testes , Fatores de Tempo , Água , QuímicaRESUMO
Simplified interspecies nuclear transfer in mouse and bovine and the possibilities of the embryos reprogramming were developed. The zona and nucleus of the bovine oocyte were removed by pronase and bisection method respectively. When the skin fibroblast of mouse was fused with the nucleus-free oocyte of the bovine using 40?s and 1. 5 kv/cm direct current pulse, the rates of fusion and cleavage were 67. 44% and 30.23% respectively. Reconstructed heterogenous embryos finally developed to 8-cell stage when they were previously activated by ionomycin and 6-DMAP and subsequently cultured in press-made micro-well in the drop of the medium. Zona-free bisection method could be used for mouse heterogenous embryos cloning, and the micro well culture method could be used to avoid zona-free embryos aggregation.