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This method is used to inexpensively prepare home-made competent cells of E. coli. The transformation efficiency is at the high end of chemical-efficient competent cells, and close to library-efficient competent cells.

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[Bio101] RbCl Super Competent Cells
[Bio101] RbCl 超级感受态细胞的制备

微生物学 > 微生物细胞生物学 > 细胞分离和培养
作者: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
6/5/2011, 11219 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.76

[Abstract] This method is used to inexpensively prepare home-made competent cells of E. coli. The transformation efficiency is at the high end of chemical-efficient competent cells, and close to library-efficient competent cells.
Keywords: Competent cells(感受态细胞), E coli(大肠杆菌), Chemical competent cells(化学活性细胞), Rubidium chloride(氯化铷)

[Abstract] 该方法介绍怎样较低廉的自己制备感受态E. coli细胞,其转化效率高于化学性的感受态细胞,可与文库感受态细胞相近。

Materials and Reagents

  1. 3 mM hexamine cobalt chloride
  2. Tryptone 
  3. Yeast extract
  4. NaCl
  5. DMSO
  6. KCl
  7. MgCl2
  8. MgSO4
  9. KOH
  10. CaCl2.2H2O
  11. RbCl
  12. SOB or 2x YT (see Recipes)
  13. MES (see Recipes)
  14. TFB (50 ml) enough for 80 tubes/400 transformations. DMSO (see Recipes)

Equipment

  1. Microcentrifuge

Procedure

  1. Inoculate overnight culture at room temperature (RT) in 5 ml SOB or 2x YT.
  2. Add overnight to 500 ml of SOB or 2x YT in 4 L flask to maximize aeration. 
  3. Add 36 ml of 5 M NaCl. Shake well at 30 °C.
  4. Grow to OD600 of 0.5. About 3 h.
  5. Spin cells. Drain pellet very well. Suck out extra liquid.
  6. Gently resuspend in 50 ml of cold TFB. Incubate for 15 min on ice.
  7. Prepare a dry ice/ethanol bath (should be thick in consistency) or liquid nitrogen.
  8. While swirling cells add 1.75 ml DMSO (no need to sterilize DMSO).
  9. Incubate cells for 10 min on ice and chill eppendorfs on ice.
  10. Add 0.5 ml cells to each Eppendorf and quickly drop tubes into dry ice bath. Store cells at -70 °C for up to 12 months.
  11. Use 100 μl for transformation. Heat shock at 42 °C for 1.5 to 2 min.

Recipes

  1. SOB or 2x YT (1 L)
    Tryptone             20 g
    Yeast extract       5 g
    5 M NaCl             2 ml
    pH to 7.0 and bring up to 1 L
    Autoclave and add 2.5 ml 1 M KCl, 10 ml of 1 M MgCl2, 10 ml 1 M MgSO4
  2. MES
    1 M MES solution made to pH 6.2 with KOH
    Filter sterilized and stored frozen
  3. TFB (50 ml) enough for 80 tubes/400 transformations
    0.5 ml of 1 M MES
    0.6 g of RbCl (makes 100 mM final conc.)
    0.45 g MnCl2 (45 mM)
    0.077 g CaCl2.2H2O (10 mM)
    0.04 g hexamine cobalt chloride (3 mM)
    Store at 4 °C
    Keeps for several months
    Use it cold

References

  1. http://sinclairfs.med.harvard.edu/methods/supercompetentecoli.php

材料与试剂

 

1.        SOB or 2xYT

2.        MES

3.        足够80 个管子的TFB (50 mL) /400转化DMSO

4.        5 M NaCl

 

仪器

 

1.        离心机

 

步骤

 

1.        室温下在5 ml SOB2xYT中接种菌并且培养过夜。

2.        加过夜培养的菌到500 ml SOB 2xYT,其装在4L瓶中以最大限度的透气。加入36 ml 5 M NaCl,在30度下培养。

3.        大约3hOD600 达到0.5

4.        离心细胞。取沉淀,吸取多余的液体。

5.        50mlsTFB重悬细胞,冰上孵育15min

6.        准备干冰/乙醇浴(厚度一直)或液氮。

7.        当离心细胞时,加入1.75ml DMSO。(DMSO不需要灭菌)

8.        冰上孵育细胞10 min,并且在冰上冷冻离心管。

9.        每个离心管加0.5 ml细胞,迅速将比例管放置于干冰上,细胞在-70°C可以达12个月。

10.    使用100 ul转化。42°C热激1.52 min.

 

配方

 

1.        SOB or 2xYT (1 L)

胰蛋白胨 20g

酵母提取物 5g

5M NaCl 2 ml

pH调到7.0  定容到1L

高压灭菌,加2.5 ml 1 M KCl, 10 ml of 1 M MgCl2, 10 ml 1M MgSO4

2.        MES

KOH1 M MES 溶液pH6.2 ,过滤灭菌,冷冻储藏。

3.        TFB (50 mL) 足够80/400 转化子使用

0.5 ml of 1M MES
0.6 g of RbCl (makes 100 mM final conc.)
0.45 g MnCl2 (45 mM)
0.077 g CaCl2.2H20 (10 mM)
0.04 g
二甲基四胺氯化钴 (3 mM)
4℃
储藏,保存几个月,低温使用

 

参考文献

 

1.        http://sinclairfs.med.harvard.edu/methods/supercompetentecoli.php

 

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How to cite this protocol: Li, X. (2011). RbCl Super Competent Cells. Bio-protocol Bio101: e76. DOI: 10.21769/BioProtoc.76; Full Text



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12/6/2014 4:39:08 AM  

Steven Cummings
Ohio State University

Hello,

I tried to follow the link in your references section but could not get it to work. Do you by any chance have a different link?

Thanks a lot!
Steven

12/6/2014 7:47:04 AM  

Xiyan Li (Author)
Department of Genetics, Stanford University, USA

Sorry, I don't have it.
You may ask the PI if you really want an original copy.

Reply

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5/27/2014 2:26:50 AM  

Hongjiu Dai
Genscript

when we prepare some vectors, some fragments have toxin genes and sequences. My may be hard to get vector amplification in E.coli. Do you think your super competent cells could solve this problem, thanks

5/27/2014 8:11:00 AM  

Xiyan Li (Author)
Department of Genetics, Stanford University, USA

No, i won't help.

What you need is a suitable host strain that us more tolerant.

I would also try low copy number plasmid and low culture temperature, e.g. 30C or even 25C.

Reply

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