Competent Cell Preparation

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!