今日之森

还有多远,一念之间。

基因克隆

2021-09-23


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

  1. 切DNA条带放入1.5mL管中,+结合液DB没过胶条即可;
  2. 放入65℃水浴或烘箱10min,待凝胶全部融化;
  3. 倒入吸附柱AC中,静置2min,12000rpm,30s,倒废液;
  4. 加800μL WB,12000rpm,30s,倒废液;
  5. 加800μL WB,12000rpm,30s,倒废液;
  6. 空转12000rpm,2min;
  7. 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

  1. 摇菌过夜,8000rpm,3min;
  2. +250μL solution I(RnaseA),涡旋震荡;
  3. +250μL solution II,轻轻颠倒混匀4-6次,反应4min;
  4. +350μL solution III,颠倒混匀至白色沉淀,12000rpm,10min;
  5. 上清液转至2mL HiBind DNA结合柱中,12000rpm,15s,弃掉废液;
  6. +500μL HBC Buffer, 12000rpm,15s;
  7. +700μL DNA Wash Buffer,12000rpm,15s,重复一次;
  8. 12000rpm,空转2min;
  9. 烘5min-10min,同时烘Elution buffer;
  10. 柱子转至新的1.5mL离心管中,+50μL Elution buffer,静置2min,12000rpm,离心1min.