1 常用载体抗性
载体 |
抗性 |
4T-1 |
Amp |
AD |
Amp |
BD |
K |
GHA |
K |
pvx |
K |
2 胶回收步骤
- 切DNA条带放入1.5mL管中,+结合液DB没过胶条即可;
- 放入65℃水浴或烘箱10min,待凝胶全部融化;
- 倒入吸附柱AC中,静置2min,12000rpm,30s,倒废液;
- 加800μL WB,12000rpm,30s,倒废液;
- 加800μL WB,12000rpm,30s,倒废液;
- 空转12000rpm,2min;
- 取出吸附柱AC,放入新的1.5mL管中,+35μL EB,静置2min,12000rpm,2min.
3 质粒提取
- 摇菌过夜,8000rpm,3min;
- +250μL solution I(RnaseA),涡旋震荡;
- +250μL solution II,轻轻颠倒混匀4-6次,反应4min;
- +350μL solution III,颠倒混匀至白色沉淀,12000rpm,10min;
- 上清液转至2mL HiBind DNA结合柱中,12000rpm,15s,弃掉废液;
- +500μL HBC Buffer, 12000rpm,15s;
- +700μL DNA Wash Buffer,12000rpm,15s,重复一次;
- 12000rpm,空转2min;
- 烘5min-10min,同时烘Elution buffer;
- 柱子转至新的1.5mL离心管中,+50μL Elution buffer,静置2min,12000rpm,离心1min.