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Competent Cell Preparation

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1040
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: 
~undefined0.5% yeast extract 
~undefined2% tryptone 
~undefined10mM NaCl(0.581g/L)
~undefined2.5mM KCl(0.191g/L)
~undefined10mM MgCl 2
~undefined10mM MgSO 4  
~undefinedDissolve in nanopure water and autoclave to sterilize. 

2. TB solution: 
~undefined10mM PIPES 
~undefined15mM CaCl 2 (1.66g/L)
~undefined250mM KCl(18.6g/L)
~undefinedDissolve in nanopure water and adjust pH to 6.7 with KOH or HCl and then add MnCl 2  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  细胞实验技术论坛   http://bbs.bbioo.com/forum-138-1.html

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. 
~undefinedNote: 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!
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