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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, 11674 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

材料和试剂

  1. 3mM六胺氯化钴
  2. 胰蛋白胨
  3. 酵母抽提物
  4. NaCl
  5. DMSO
  6. KCl
  7. MgCl 2
  8. MgSO 4
  9. KOH
  10. CaCl 2 2·2H 2 O
  11. RbCl
  12. SOB或2x YT(参见配方)
  13. MES(见配方)
  14. TFB(50ml),足以进行80管/400转化。 DMSO(见配方)

设备

  1. 微量离心机

程序

  1. 在室温(RT)下在5ml SOB或2x YT中接种过夜培养物。
  2. 在4L烧瓶中加入500ml SOB或2x YT过夜,以最大限度地增加曝气量。
  3. 加入36ml 5M NaCl。 在30℃振荡。
  4. 生长至OD 600的0.5。 约3小时。
  5. 旋转细胞。 排干沉淀非常好。 吸出多余的液体。
  6. 轻轻地重悬在50毫升冷TFB。 在冰上孵育15分钟。
  7. 准备干冰/乙醇浴(应稠厚)或液氮。
  8. 旋转细胞加入1.75ml DMSO(不需要灭菌DMSO)。
  9. 孵育细胞在冰上10分钟,冰上冷却eppendorf。
  10. 向每个Eppendorf中加入0.5ml细胞,并迅速将管子放入干冰浴中。 将细胞储存在-70°C长达12个月。
  11. 使用100μl转化。 在42℃热冲击1.5至2分钟。

食谱

  1. SOB或2x YT(1 L)
    胰蛋白胨               20克
    酵母提取物        5克
    5 M NaCl              2 ml
    pH至7.0,并升至1 L
    高压灭菌并加入2.5ml 1M KCl,10ml 1M MgCl 2,10ml 1M MgSO 4。
  2. MES
    1M MES溶液用KOH调至pH6.2 过滤灭菌并冷冻保存
  3. TFB(50ml),足以进行80管/400次转化
    0.5 ml 1M MES
    0.6g RbCl(使最终浓度为100mM)
    0.45g MnCl 2(45mM) 0.077g CaCl 2·2H 2 O(10mM)
    0.04g氯化六胺钴(3mM) 存储在4°C
    保存几个月
    使用它冷

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.

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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.

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