The bacterial cultures, which are transformed with the desired plasmid DNA grow overnight in growth media with appropriate antibiotic in a 37°C shaking incubator. strains grown and culture with anti-biotics. 10ml LB加抗生素10微升，
Amp / Kan / Chm should in 1ul:1ml 都是10ul/10ML,50ul/50ML。
1. Harvest. The next day, the bacteria culture 5ml is pelleted by centrifugation 1min ,13200rpm and the supernatant is removed. 标号码, 养过夜之后取出来 离心一次再加一次，沉淀多一些，加试剂用一支枪和枪头就好
2. Resuspend. add 250ul （1000P-025）resuspension buffer R3 with RNase to the cell pellet. The remaining pellet is resuspended in lysis buffer. and resuspend the pellet until it is homogeneous. 溶解足够用震荡仪。吸出所有残 液，用纸干净吸水。
3. Lyse. Add 250ul lysis buffer L7 mix gently by inverting the cap-tube until the mixture is homogeneous.变轻就可以不一定等5分钟 Incubate the tube at room temperature for 5 min.
4. Precipitate. Add 350ul precipitation buffer N4. （1000P-035）立即马上混匀 因为沉淀物凝集Mix immediately by inverting the tube vigorously shaking the tube until the mixture is homogeneous. Then centrifuge the lysate at 12000g for 10 min.准备好过滤网和套管，标号码，过滤后留液体。
5. Binding. Load the supernatant on to a spin column in a 2ml wash tube. centrifuge the column at 12000g for 1 min. discard the flow-through and place column back into the wash tube.标号码
6. Wash. add 500ul wash buffer W10 with ethanol to the column. （1000P-050） 可以不用这步看具体实验incubate the column for 1 min at room temperature. Centrifuge the column at 12000g for 1min. discard the flow-through and place column back into the wash tube
7. Wash and remove ethanol. Add 700ul（1000P-070） wash buffer W9直接用 这步 with ethanol to the column. 放在冰上一会Centrifuge the column at 12000g for 1min. discard the flow-through and place column back into the wash tube.倒掉液体再离心一次，保持干燥过滤网。用纸干净吸水，换新管， 标号码，准备TE加热66度。
8. Elute. Place the spin column in a clean 1.5 ml recovery tube Add 50ul （200P-050）of preheated TE buffer to the center of the column. Incubate the column for 1 min at room temperature. 换新管，过滤网放上标号码，在中间加 入已经加温。 Centrifuge the column at 12000g for 2 min. Discard the column, store plasmid DNA at 4 o C. 取出过滤网留液体， 准备好跑胶
9. minipreps- plasmid using Invitrogen Plasmid Miniprep kits