搜索

[Bio101] A Quick and Easy Method for Making Competent Escherichia coli Cells for Transformation Using Rubidium Chloride
[Bio101] 一种使用氯化铷快捷简易制备进行细胞转化的大肠埃希杆菌感受态的方法   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

This protocol describes a quick and efficient method to make competent E. coli cells for transformation using rubidium chloride. Commercial competent cells are expensive and this protocol provides a cheaper alternative to them.

Keywords: Competent cells(感受态细胞), E. coli(大肠埃希杆菌), Transformation efficiency(转化效率), TOP10(TOP10), DH5α(DH5α)

Background

The success of gene cloning is highly dependent on the transformation efficiency of bacterial cells. The efficiency can be artificially improved by treating the cells with chemicals or electric pulses. Several protocols are available to prepare competent E. coli cells, however, they are usually long, laborious, and show inconsistency in competence. The protocol by Green and Rogers (2013) overcomes these downsides and allows the preparation of highly competent cells (~106-108 CFU/µg DNA). While other protocols require cells to be grown at low temperature (19-22 °C), this protocol involves growing cells at 37 °C. Thus, the cells grow faster and reach log phase within 4 h as compared to 18-24 h. This protocol is highly reproducible.

Materials and Reagents

  1. 25 mm, 0.2 µm syringe filter PES (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 725-2520 )
  2. 5 ml syringe (Fisher Scientific, catalog number: 14-829-45)
    Manufacturer: BD, catalog number: 309646 .
  3. 1.5 ml Eppendorf tubes (Corning, Costar®, catalog number: 3207 )
  4. 100 mm Petri dish (Corning, Falcon®, catalog number: 351029 )
  5. Pipettes tips
  6. 10 ml culture tubes (VWR, catalog number: 60818-725 )
  7. Spreaders (Fisher Scientific, catalog number: 14-665-230 )
  8. 250 ml centrifuge bottles (Sigma-Aldrich, catalog number: Z353736)
    Manufacturer: Thermo Fisher Scientific, catalog number: 3141-0250 .
  9. E. coli strain (TOP10 or DH5α)
  10. Control plasmid (e.g., pUC19)
  11. LB broth (Fisher Scientific, catalog number: BP9723-500 )
  12. LB agar (BD, catalog number: 244510 )
  13. Ice
  14. Liquid nitrogen
  15. Rubidium chloride (RbCl) (Sigma-Aldrich, catalog number: R2252 )
  16. Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Sigma-Aldrich, catalog number: 203734 )
  17. Potassium acetate (Sigma-Aldrich, catalog number: P1190 )
  18. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C8106 )
  19. Glycerol (Fisher Scientific, catalog number: BP229-4 )
  20. MOPS (Fisher Scientific, catalog number: BP308-100 )
  21. Acetic acid (Fisher Scientific, catalog number: A38-212 )
  22. Sodium hydroxide (Fisher Scientific, catalog number: S318-1 )
  23. Vector specific Antibiotics
  24. Buffer I (see Recipes)
  25. Buffer II (see Recipes)

Equipment

  1. Pipettes (P1000 and P200)
  2. 1 L flasks (Fisher Scientific, catalog number: 10-040K)
    Manufacturer: Corning, catalog number: C49801L .
  3. Spectrophotometer (Beckman Coulter, model: DU-640 , catalog number: 8043-30-1090)
  4. Water bath (Marshall Scientific, model: Precision 181 )
  5. 37 °C incubator and shaker (Thermo Fisher Scientific, model: MaxQTM 4000 , catalog number: SHKE4000-1CE)
  6. Centrifuge (Thermo Fisher Scientific, SorvallTM, model: RC-5B , catalog number: 8327-30-1004)
  7. pH meter (Fisher Scientific, catalog number: 13-644-928)
    Manufacturer: Thermo Fisher Scientific, catalog number: 1112106 .

Procedure

  1. Prepare competent cells
    Note: All steps are carried out in sterile conditions.
    1. Day 1 (1 h preparation + overnight)
      Streak E. coli strain (e.g., TOP10 or DH5α) on an LB agar plate in a 3 streak fashion (Reference 2) and incubate overnight at 37 °C.
    2. Day 2 (10 min preparation + overnight)
      Pick a single E. coli colony and culture in 5 ml LB broth in a 10 ml culture tube at 37 °C overnight at 200 RPM.
    3. Day 3 (7 h)
      1. Inoculate 500 ml LB broth in a 1 L flask with 500 µl overnight E. coli culture and grow at 37 °C (200 RPM) until OD600 = 0.5 (about 4 h).
      2. Chill cells on ice for 10 min.
      3. Centrifuge E. coli cells at 2,000 x g for 10 min at 4 °C.
      4. Remove supernatant and resuspend the pellet in 50 ml ice-cold buffer I (see Recipes). Keep the cells on ice for the entire time and shake manually in circular motions on ice. It will take 20-30 min to resuspend the cells (Video 1).
        Note: Do not pipette the cells up and down, but you may break the pellet with a pipette for faster resuspension.
      5. Centrifuge cells at 2,000 x g for 10 min at 4 °C.
      6. Remove supernatant and resuspend the pellet in 25 ml ice-cold buffer II (see Recipes) by shaking. Keep the cells on ice for the entire time. It will take some time (20-30 min) to resuspend the cells (Video 1).
        Note: Do not pipette the cells up and down, but you may break the pellet with a pipette for faster resuspension.

        Video 1. Resuspending pellet in buffer I or buffer II: Gently rotate bottles in an ice-bucket to resuspend the bacterial pellet in buffer

      7. Aliquot 50 μl cells into pre-chilled Eppendorf tubes on ice and immediately freeze the tubes in liquid nitrogen. Work quickly through this step.
      8. Store competent cells at -80 °C.

  2. Check competence of cells
    1. Day 1 (2 h preparation +overnight)
      1. Thaw two tubes of competent cells on ice for 5 min.
      2. Add 1 ng positive control plasmid (pUC19) to one tube and 1 μl water to the other tube as a negative control.
      3. Incubate on ice for 30 min.
      4. Heat-shock the cells at 42 °C in a water bath for 30 sec and immediately put the tubes on ice for 2 min.
      5. Add 200 μl LB broth to the cells and grow the cells at 37 °C for one hour at 200 RPM.
        Note: Fresh LB is not necessary at this step, however you must use uncontaminated LB.
      6. Plate 50 μl (1/5th of total volume) on an LB agar plate with appropriate antibiotic.
      7. Grow the cells overnight at 37 °C.
    2. Day 2 (30 min)
      Check the efficiency of competent cells by counting the number of colonies on both positive and negative control.
      Note: If the transformation efficiency is high and there is a high number of evenly spread colonies, you may count colonies on a fourth of the plate area and multiply the value by 4 for an approximate total number of colonies. Else, you may start with a lower concentration of control plasmid (1 pg-1 ng). The negative control should have no colonies as any colonies found indicate contamination with an antibiotic-resistant strain.

Notes

This protocol is highly reproducible if you:

  1. Keep the cells on ice at all times and work fast through the process.
  2. Prepare buffers fresh on the day of the competent cells preparation.
  3. Work with small culture volumes (e.g., 200 ml) to shorten the duration of the preparation.

Recipes

  1. Buffer I (prepare 50 ml per 500 ml culture)
    Final concentrations:
    100 mM RbCl (12 g/L)
    50 mM MnCl2·4H2O (9.89 g/L)
    30 mM potassium acetate (2.94 g/L)
    10 mM CaCl2·2H2O (1.47 g/L)
    15% (v/v) glycerol (150 ml/L)
    Adjust pH to 5.8 with dilute acetic acid and filter sterilize
    Note: Store at 4 °C, protect from light.
  2. Buffer II (prepare 25 ml per 500 ml culture)
    Final concentrations:
    10 mM MOPS (2.09 g/L)
    10 mM RbCl (1.2 g/L)
    75 mM CaCl2·2H2O (11 g/L)
    15% (v/v) glycerol (150 ml/L)
    Adjust pH to 6.5 with dilute NaOH and filter sterilize
    Note: Store at 4 °C and protect from light.

Acknowledgments

This protocol is adapted from Green and Rogers (2013). I would like to thank Bergmann lab members for fine-tuning this protocol.
The authors declare that there are no conflicts of interest.

References

  1. Green, R. and Rogers, E. J. (2013). Chemical transformation of E. coli. Methods Enzymol 529: 329-336.
  2. Streaking and Isolating Bacteria on an LB Agar Plate (https://www.addgene.org/protocols/streak-plate/)

简介

这个协议描述了一个快速有效的方法来做出胜任的E。 使用氯化铷转化大肠杆菌细胞。 商业感受态细胞是昂贵的,这个协议提供了一个更便宜的选择。

【背景】基因克隆的成功高度依赖于细菌细胞的转化效率。 通过用化学物质或电脉冲处理细胞可以人为地提高效率。 有几个协议可用于准备主管的E。 然而,它们通常是长期的,费力的,并且在能力上表现出不一致。 Green和Rogers(2013)的方案克服了这些缺点,并允许制备高感受态细胞(〜10 6 -10 8 CFU /μgDNA)。 虽然其他协议要求细胞在低温(19-22°C)下生长,但该协议涉及在37°C下生长细胞。 因此,与18-24小时相比,细胞生长更快并且在4小时内达到对数期。 该协议是高度可重复的。

关键字:感受态细胞, 大肠埃希杆菌, 转化效率, TOP10, DH5α

材料和试剂

  1. 25毫米,0.2微米针筒式过滤器PES(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:725-2520)
  2. 5毫升注射器(Fisher Scientific,目录号:14-829-45)
    制造商:BD,目录号:309646。
  3. 1.5ml Eppendorf管(Corning,Costar ,产品目录号:3207)
  4. 100毫米培养皿(Corning,Falcon ,目录号:351029)
  5. 移液器提示
  6. 10毫升培养管(VWR,目录号:60818-725)
  7. 撒肥机(Fisher Scientific,目录号:14-665-230)
  8. 250毫升离心瓶(西格玛奥德里奇,目录号:Z353736)
    制造商:Thermo Fisher Scientific,目录号:3141-0250。
  9. 电子。大肠杆菌菌株(TOP10或DH5α)
  10. 对照质粒(例如,pUC19)
  11. LB肉汤(Fisher Scientific,目录号:BP9723-500)
  12. LB琼脂(BD,目录号:244510)

  13. 液氮
  14. 氯化铷(RbCl)(Sigma-Aldrich,目录号:R2252)
  15. 氯化锰(II)四水合物(MnCl 2•4H 2 O)(Sigma-Aldrich,目录号:203734)
  16. 乙酸钾(Sigma-Aldrich,目录号:P1190)
  17. 氯化钙二水合物(CaCl 2•2H 2 O)(Sigma-Aldrich,目录号:C8106)
  18. 甘油(Fisher Scientific,目录号:BP229-4)
  19. MOPS(Fisher Scientific,目录号:BP308-100)
  20. 乙酸(Fisher Scientific,目录号:A38-212)
  21. 氢氧化钠(Fisher Scientific,目录号:S318-1)
  22. 载体特异性抗生素
  23. 缓冲区I(见食谱)
  24. 缓冲区二(见食谱)
  • 第二天(30分钟)
    通过计数阳性和阴性对照上的菌落数来检查感受态细胞的效率。
    注意:如果转化效率高,并且有大量均匀扩散的菌落,则可以在平板区域的四分之一上计数菌落,并将该值乘以大约4的菌落总数。否则,您可以从较低浓度的对照质粒(1pg-1ng)开始。阴性对照应该没有菌落,因为任何发现的菌落都表明污染了抗生素抗性菌株。
  • 笔记

    这个协议是高度重现的,如果你:


    1. 任何时候都要将细胞放在冰上,并在整个过程中快速工作
    2. 在感受态细胞制备当天准备新鲜的缓冲液。
    3. 使用少量的培养物(例如,200毫升)来缩短准备时间。

    食谱

    1. 缓冲液I(每500ml培养物准备50ml)
      最终浓度:
      100毫克RbCl(12克/升)
      50mM MnCl 2•4H 2 O(9.89g / L)
      30mM乙酸钾(2.94g / L)
      10mM CaCl 2•2H 2 O(1.47g / L)
      15%(v / v)甘油(150ml / L)
      用稀乙酸调节pH值至5.8,过滤灭菌 注意:储存在4°C避光保存。
    2. 缓冲液II(准备每500毫升培养25毫升)
      最终浓度:
      10 mM MOPS(2.09 g / L)
      10mM RbCl(1.2g / L)
      75mM CaCl 2•2H 2 O(11g / L)
      15%(v / v)甘油(150ml / L)
      用稀NaOH调节pH至6.5并过滤灭菌 注意:在4°C储存并避光。

    致谢

    该协议改编自Green和Rogers(2013)。 我想感谢Bergmann实验室成员对这个协议进行微调。
    作者宣称没有利益冲突。

    参考

    1. Green,R.和Rogers,E. J.(2013)。 化学转化 大肠杆菌。方法Enzymol 529:329-336。
    2. 在LB琼脂平板上划线和分离细菌( https://www.addgene.org/protocols/ 条纹板/
    • English
    • 中文翻译
    免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
    Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
    引用:Sharma, N., Gil, M. A. and Wengier, D. (2017). A Quick and Easy Method for Making Competent Escherichia coli Cells for Transformation Using Rubidium Chloride. Bio-protocol Bio101: e2590. DOI: 10.21769/BioProtoc.2590;
    提问与回复

    (提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

    当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。