CompetentCellPreparation

实验概要

Competent cells are those that possess more easily altered cell walls that DNA can be passed through easily. These cells readily incorporate foreign DNA. On example of a competent cell is E. coli.

主要试剂

1. SOB solution:
*0.5% yeast extract
*2% tryptone
*10mM NaCl(0.581g/L)
*2.5mM KCl(0.191g/L)
*10mM MgCl₂
*10mM MgSO₄
*Dissolve in nanopure water and autoclave to sterilize.

2. TB solution:
*10mM PIPES
*15mM CaCl₂(1.66g/L)
*250mM KCl(18.6g/L)
*Dissolve in nanopure water and adjust pH to 6.7 with KOH or HCl and then add MnCl₂ to 55mM (10.88g/L), and adjust to final volume. Sterilize by
filtration with 0.45um filter and store at 4°C

实验步骤

1. Culture cells (DH5a in my case) on LB agar plate at 37°Covernight.
2. Pick up 10 -12 large colonies and culture in 250ml SOB in a 1L flask, 19°C with vigorous shaking to OD600=0.5 (normally it takes 24-36hrs)
3. Place the flask in ice for 10 min.
4. Pelleting the cell by spining at 4000rpm for 10 min at 4°C.
5. Gently resuspend the cell in 80ml ice-cold TB and store on ice for 10 min.
6. Spin at 4000rpm for 10 min at 4°C .
7. Gently resuspend the pellet in 20ml ice-cold TB and 1.4ml DMSO (the DMSO needs to be stored at -20°C o/n before use).
8. Aliquote the cell to 50 to 500ul for transformation or store at -70°C.
*Note: The E. coli cells prepared this way are normally 100 to 1000 times more efficient than normal calcium method, so do not plate too dense!