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[Bio101] The Inoue Method for Preparation and Transformation of Competent E. coli: "Ultra Competent" Cells
[Bio101] Inoue法制备和转化大肠杆菌超级感受态细胞   

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Abstract

This protocol differs from other procedures in that the bacterial culture is grown at 18 °C rather than the conventional 37 °C. Otherwise, the protocol is unremarkable and follows a fairly standard course. Why growing the cells at low temperature should affect the efficiency of transformation is unknown. Perhaps the composition or the physical characteristics of bacterial membranes synthesized at 18 °C are more favorable for uptake of DNA, or perhaps the phases of the growth cycle that favor efficient transformation are extended. Incubating bacterial cultures at 18 °C is a challenge. Most laboratories do not have a shaking incubator that can accurately maintain a temperature of 18 °C summer and winter. One solution is to place an incubator in a 4 °C cold room and use the temperature control to heat the incubator to 18 °C. Alternatively, there is almost no loss of efficiency if the cultures are grown at 20-23 °C, which is the ambient temperature in many laboratories. Cultures incubated at these temperatures grow slowly with a doubling time of 2.5 to 4 h. To avoid reaching desired OD late at night, set up cultures in the evening and harvest the bacteria early the following morning. The procedure works well with many strains of E. coli in common use in molecular cloning, including XL1-Blue, DH1, JM103, JM108/9, DH5a, and HB101.

Materials and Reagents

  1. Strains of E. coli: XL1-Blue, DH1, JM103, JM108/9, DH5a, and HB101.
  2. DMSO: Oxidation products of DMSO, presumably dimethyl sulfone and dimethyl sulfide, are inhibitors of transformation (Hanahan, 1985). To avoid problems, purchase DMSO of the highest quality. (Merck KGaA/EMD Millipore, catalog number: ES-002-10F )
  3. PIPES (Alfa Aesar, catalog number: 3p B21835-22 )
  4. Deionized H2O
  5. Yeast Extract
  6. Tryptone
  7. KCl
  8. NaCl
  9. NaOH
  10. MgCl2
  11. MgSO4
  12. Antibiotic
  13. MnCl2.4 H2O
  14. CaCl2.2 H2O
  15. Glucose
  16. Liquid nitrogen
  17. Ethanol
  18. Sugar
  19. Inoue transformation buffer (see Recipes)
  20. SOB medium (see Recipes)
  21. SOC medium (see Recipes)
  22. Luria-Bertani (LB) medium (see Recipes)
  23. 0.5 M piperazine-1,2-bis(2-ethanesulfonic acid) (PIPES) (pH 6.7) (see Recipes)

Equipment

  1. Centrifuges and Rotors (American Laboratory Trading)
  2. Milli-Q filtration system (EMD Millipore)
  3. Polypropylene 2059 tubes (17 x 100 mm) (BD Biosciences, Falcon®), chilled in ice
  4. Shaking Incubator (18 °C)
  5. Water bath (42 °C)
  6. Nalgene filter
  7. Disposable prerinsed Nalgene filter (0.45-mm pore size)
  8. 250-ml flask
  9. Sorvall GSA rotor
  10. Vacuum aspirator
  11. Bent glass rod
  12. Bunsen burner
  13. 0.22 μm filter

Procedure

Note: all steps in this protocol should be carried out aseptically

  1. Preparation of cells
    1. Prepare Inoue transformation buffer (chilled to 0 °C before use). Organic contaminants in the H2O used to prepare transformation buffers can reduce the efficiency of transformation of competent bacteria. H2O obtained directly from a well-serviced Milli-Q filtration system usually gives good results. If problems should arise, treat the deionized H2O with activated charcoal before use.
      1. Prepare 0.5 M PIPES (pH 6.7). Adjust the pH of the solution to 6.7 with 5 M KOH, and then add pure H2O to bring the final volume to 100 ml. Sterilize the solution by filtration through a disposable prerinsed Nalgene filter. Divide into aliquots and store frozen at -20 °C
      2. Prepare Inoue transformation buffer by dissolving all of the solutes listed below in 800 ml of pure H2O and then add 20 ml of 0.5 M PIPES (pH 6.7). Adjust the volume of the Inoue transformation buffer to 1 liter with pure H2O.
      3. Sterilize Inoue transformation buffer by filtration through a prerinsed 0.45-mm Nalgene filter. Divide into aliquots and store at -20 °C.
    2. Pick a single bacterial colony (2-3 mm in diameter) from a plate that has been incubated for 16-20 h at 37 °C. Transfer the colony into 25 ml of LB broth or SOB medium in a 250 ml flask. Incubate the culture for 6-8 h at 37 °C with vigorous shaking (250-300 rpm).
    3. At about 6 o'clock in the evening, use this starter culture to inoculate three 1-L flasks, each containing 250 ml of SOB. The first flask receives 10 ml of starter culture, the second receives 4 ml, and the third receives 2 ml. Incubate all three flasks overnight at 18-22 °C with moderate shaking.
    4. The following morning, read the OD600 of all three cultures. Continue to monitor the OD every 45 min.
    5. When the OD600 of one of the cultures reaches 0.55, transfer the culture vessel to an ice-water bath for 10 min. Discard the two other cultures.
    6. The ambient temperature of most laboratories rises during the day and falls during the night. The number of degrees and the timing of the drop from peak to trough varies depending on the time of year, the number of people working in the laboratory at night, and so on. Because of this variability, it is difficult to predict the rate at which cultures will grow on any given night. Using three different inocula increases the chances that one of the cultures will be at the correct density after an overnight incubation.
    7. Harvest the cells by centrifugation at 2,500 x g (3,900 rpm in a Sorvall GSA rotor) for 10 min at 4 °C.
    8. Pour off the medium and store the open centrifuge bottle on a stack of paper towels for 2 min. Use a vacuum aspirator to remove any drops of remaining medium adhering to walls of the centrifuge bottle or trapped in its neck.
    9. Resuspend the cells gently in 80 ml of ice-cold Inoue transformation buffer. The cells are best suspended by swirling rather than pipetting or vortexing.
    10. Harvest the cells by centrifugation at 2,500 x g (3,900 rpm in a Sorvall GSA rotor) for 10 min at 4 °C.
    11. Pour off the medium and store the open centrifuge tube on a stack of paper towels for 2 min.
    12. Use a vacuum aspirator to remove any drops of remaining medium adhering to the walls of the centrifuge tube or trapped in its neck.

  2. Freezing of competent cells
    1. Resuspend the cells gently in 20 ml of ice-cold Inoue transformation buffer.
    2. Add 1.5 ml of DMSO. Mix the bacterial suspension by swirling and then store it in ice for 10 min.
    3. Working quickly, dispense aliquots of the suspensions into chilled, sterile microfuge tubes.
    4. Immediately snap-freeze the competent cells by immersing the tightly closed tubes in a bath of liquid nitrogen. Store the tubes at -70 °C until needed. Freezing in liquid nitrogen enhances transformation efficiency by ~5-fold. For most cloning purposes, 50 ml aliquots of the competent-cell suspension will be more than adequate. However, when large numbers of transformed colonies are required (e.g., when constructing cDNA libraries), larger aliquots may be necessary.
    5. When needed, remove a tube of competent cells from the -70 °C freezer. Thaw the cells by holding the tube in the palm of the hand. Just as the cells thaw, transfer the tube to an ice bath. Store the cells on ice for 10 min.
    6. Use a chilled, sterile pipette tip to transfer the competent cells to chilled, sterile 17 x 100-mm polypropylene tubes. Store the cells on ice. Glass tubes should not be used since they lower the efficiency of transformation by ~10-fold.

  3. Transformation
    1. Include all of the appropriate positive and negative controls.
    2. Add the transforming DNA (up to 25 ng per 50 ml of competent cells) in a volume not exceeding 5% of that of the competent cells. Swirl the tubes gently several times to mix their contents. Set up at least two control tubes for each transformation experiment, including a tube of competent bacteria that receives a known amount of a standard preparation of superhelical plasmid DNA and a tube of cells that receives no plasmid DNA at all. Store the tubes on ice for 30 min.
    3. Transfer the tubes to a rack placed in a preheated 42 °C circulating water bath. Store the tubes in the rack for exactly 90 sec. Do not shake the tubes. Heat shock is a crucial step. It is very important that the cells be raised to exactly the right temperature at the correct rate. The incubation times and temperatures given here have been worked out using Falcon 2059 tubes. Other types of tubes will not necessarily yield equivalent results.
    4. Rapidly transfer the tubes to an ice bath. Allow the cells to cool for 1-2 min.
    5. Add 800 ml of SOC medium to each tube. Warm the cultures to 37 °C in a water bath, and then transfer the tubes to a shaking incubator set at 37 °C. Incubate the cultures for 45 min to allow the bacteria to recover and to express the antibiotic resistance marker encoded by the plasmid. To maximize the efficiency of transformation, gently agitate (<225 cycles/minute) the cells during the recovery period.
    6. Transfer the appropriate volume (up to 200 ml per 90 mm plate) of transformed competent cells onto agar SOB medium containing 20 mM MgSO4 and the appropriate antibiotic. When selecting for resistance to tetracycline, the entire transformation mixture may be spread on a single plate (or plated in top agar). In this case, collect the bacteria by centrifuging for 20 sec at room temperature (RT) in a microfuge, and then gently resuspend the cell pellet in 100 ml of SOC medium by tapping the sides of the tube. IMPORTANT Sterilize a bent glass rod by dipping it into ethanol and then in the flame of a Bunsen burner. When the rod has cooled to RT, spread the transformed cells gently over the surface of the agar plate. When selecting for resistance to ampicillin, transformed cells should be plated at low density (<104 colonies per 90 mm plate), and the plates should not be incubated for more than 20 h at 37 °C. The enzyme b-lactamase is secreted into the medium from ampicillin-resistant transformants and can rapidly inactivate the antibiotic in regions surrounding the colonies. Thus, plating cells at high density or incubating them for long periods of time results in the appearance of ampicillin-sensitive satellite colonies. This problem is ameliorated, but not completely eliminated, by using carbenicillin rather than ampicillin in selective media and increasing the concentration of antibiotic from 60 mg/ml to 100 mg/ml. The number of ampicillin-resistant colonies does not increase in linear proportion to the number of cells applied to the plate, perhaps because of growth-inhibiting substances released from the cells killed by the antibiotic.
    7. Store the plates at RT until the liquid has been absorbed.
    8. Invert the plates and incubate them at 37 °C. Transformed colonies should appear in 12-16 h.

Recipes

  1. LB medium
    Per Liter: To 950 ml of deionized H2O, add
    Tryptone 10 g
    Yeast Extract 5 g
    NaCl 10 g
    Shake until the solutes have dissolved. Adjust the pH to 7.0 with 5 N NaOH (~0.2 ml). Adjust the volume of the solution to 1 L with deionized H2O. Sterilize by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle.
  2. SOB medium
    1. Per Liter: To 950 ml of deionized H2O , add
      Tryptone 20 g
      Yeast Extract 5 g
      NaCl 0.5 g
      Shake until the solutes have dissolved. Add 10 ml of a 250 mM solution of KCl (this solution is made by dissolving 1.86 g of KCl in 100 ml of deionized H2O). Adjust the pH of the medium to 7.0 with 5 N NaOH (~0.2 ml). Adjust the volume of the solution to 1 liter with deionized H2O. Sterilize by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle. Just before use, add 5 ml of a sterile solution of 2 M MgCl2 (this solution is made by dissolving 19 g of MgCl2 in 90 ml of deionized H2O. Adjust the volume of the solution to 100 ml with deionized H2O and sterilize by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle).
    2. SOB agar plates containing 20 mM MgSO4 and the appropriate antibiotic.
    3. Standard SOB contains 10 mM MgSO4.
    4. SOB medium, for growth of culture to be transformed.
    5. Prepare three 1-liter flasks of 250 ml each and equilibrate the medium to 18-20 °C before inoculation.
  3. Inoue transformation buffer
    Reagent
    Amount/L
    Final concentration
    MnCl2.4 H2O
    10.88 g
    55 mM
    CaCl2.2 H2O
    2.20 g
    15 mM
    KCl
    18.65 g
    250 mM
    PIPES (0.5 M, pH 6.7)
    20 ml
    10 mM
    H2O
    to 1 L


    Chilled to 0 °C before use.
  4. SOC medium
    Approximately 1 ml of this medium is needed for each transformation reaction. SOC medium is identical to SOB medium except it contains 20 mM glucose. After the SOB medium has been autoclaved, allow it to cool to 60 °C or less. Add 20 ml of a sterile 1 M solution of glucose (this solution is made by dissolving 18 g of glucose in 90 ml of deionized H2O. After the sugar has dissolved, adjust the volume of the solution to 100 ml with deionized H2O and sterilized by passing it through a 0.22 μm filter).
  5. 0.5 M PIPES (pH 6.7)
    Dissolving 15.1 g PIPES in 80 ml of pure H2O.

References

  1. Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166(4): 557-580.
  2. Hanahan, D. (1985). Techniques for transformation of E. coli. In DNA cloning: A Practical Approach (ed. D.M. Glover), vol. 1 pp. 109-135. IRL Press, Oxford, United Kingdom.
  3. Inoue, H., Nojima, H. and Okayama, H. (1990). High efficiency transformation of Escherichia coli with plasmids. Gene 96(1): 23-28.

简介

该方案与其它方法的不同之处在于细菌培养物在18℃而不是常规37℃下生长。否则,协议是不明显的,并遵循一个相当标准的课程。为什么在低温下生长细胞应该影响转化的效率是未知的。也许在18℃合成的细菌膜的组成或物理特性更有利于DNA的摄取,或者有利于有效转化的生长周期的阶段延长。在18℃孵育细菌培养物是一个挑战。大多数实验室没有可以准确保持温度在夏天和冬天18°C的摇动孵化器。一种解决方案是将培养箱放置在4℃冷室中,并使用温度控制将培养箱加热至18℃。或者,如果培养物在许多实验室中的环境温度20-23℃下生长,则几乎没有效率损失。在这些温度下温育的培养物以2.5至4小时的倍增时间缓慢生长。为了避免在晚上达到期望的OD,在晚上建立培养物并且在第二天早晨收获细菌。该程序适用于许多菌株。通常用于分子克隆,包括XL1-Blue,DH1,JM103,JM108/9,DH5a和HB101。

材料和试剂

  1. 菌株E。 大肠杆菌:XL1-Blue,DH1,JM103,JM108/9,DH5a和HB101。
  2. DMSO:DMSO(推测为二甲基砜和二甲基硫醚)的氧化产物是转化的抑制剂(Hanahan,1985)。 为了避免问题,购买最高质量的DMSO。 (Merck KGaA/EMD Millipore,目录号:ES-002-10F)
  3. PIPES(Alfa Aesar,目录号:3p B21835-22)
  4. 去离子H 2 O 2 /
  5. 酵母提取物
  6. 胰蛋白酶
  7. KCl
  8. NaCl
  9. NaOH
  10. MgCl 2
  11. MgSO 4 4 /
  12. 抗生素
  13. MnCl 2 4 H sub 2 O
  14. CaCl 2 2 H sub 2 O
  15. 葡萄糖
  16. 液氮
  17. 乙醇

  18. Inoue转换缓冲区(参见配方)
  19. SOB培养基(参见配方)
  20. SOC介质(参见配方)
  21. Luria-Bertani(LB)培养基(见配方)
  22. 0.5M哌嗪-1,2-双(2-乙磺酸)(PIPES)(pH6.7)(参见配方)

设备

  1. 离心机和转子(美国实验室贸易)
  2. Milli-Q过滤系统(EMD Millipore)
  3. 将聚丙烯2059管(17×100mm)(BD Biosciences,Falcon )在冰上冷冻
  4. 摇动培养箱(18°C)
  5. 水浴(42℃)
  6. Nalgene过滤器
  7. 一次性预冲洗Nalgene过滤器(0.45 mm孔径)
  8. 250毫升烧瓶
  9. Sorvall GSA转子
  10. 真空吸气器
  11. 弯曲玻璃棒
  12. 本生灶
  13. 0.22μm过滤器

程序

注意:此协议中的所有步骤都应无菌执行

  1. 细胞的制备
    1. 制备Inoue转化缓冲液(使用前冷却至0℃)。 H 2 O 2中的有机污染物用于制备转化缓冲液 可降低感受态细菌的转化效率。 H 2 O 直接从服务良好的Milli-Q过滤系统获得   给出了良好的效果。 如果出现问题,应处理去离子H 2 O 2 使用活性炭。
      1. 制备0.5 M PIPES(pH 6.7)。   用5M KOH调节溶液的pH至6.7,然后加入纯的 H 2 O使终体积达到100ml。 消毒溶液 通过一次性预冲洗Nalgene过滤器过滤。 分为 等分试样并在-20℃冷冻保存
      2. 准备井上转化 缓冲液通过溶解所有下面列出的溶质在800毫升纯 H 2 O,然后加入20ml 0.5M PIPES(pH 6.7)。 调节音量   Inoue转化缓冲液至1升,使用纯H 2 O
      3. 消毒 Inoue转化缓冲液通过预过筛0.45-mm过滤 Nalgene滤器。 分成等份,存储在-20°C。
    2. 选择一个 单细菌菌落(直径2-3毫米)从一个板已经 在37℃孵育16-20小时。 将菌落转移到25ml LB中 肉汤或SOB培养基在250ml烧瓶中。 孵育培养6-8小时   37℃,剧烈摇动(250-300rpm)
    3. 在约6点钟   晚上,使用这种起子培养物接种三个1-L烧瓶, 每个含有250ml的SOB。 第一个烧瓶接受10ml起子   培养,第二次接受4毫升,第三次接受2毫升。 将所有三个烧瓶在18-22℃下温和振荡孵育过夜
    4. 第二天早晨,读取所有三种培养物的OD <600>。 继续每45分钟监测一次OD
    5. 当其中一种培养物的OD 600达到0.55时, 培养皿置于冰水浴中10分钟。 丢弃另外两个 文化
    6. 大多数实验室的环境温度上升 在白天和夜间下降。 度数和   从峰到谷的下降时间根据时间而变化 年,在夜间在实验室工作的人数等 上。 由于这种变异性,很难预测速率 哪些文化将在任何给定的夜晚生长。 使用三种不同 接种增加了其中一种培养物的机会 过夜孵育后的正确密度
    7. 通过在4℃以2,500×g离心(在Sorvall GSA转子中为3,900rpm)10分钟收获细胞。
    8. 倒出介质并将打开的离心机瓶存放在堆叠上 的纸巾2分钟。 使用真空吸气器清除任何液滴   剩余介质粘附在离心瓶的壁上或被捕获 在其颈部
    9. 重悬细胞轻轻在80毫升的冰冷 Inoue转化缓冲液。 细胞最好通过涡流悬浮 而不是移液或涡旋
    10. 通过在4℃以2,500×g离心(在Sorvall GSA转子中为3,900rpm)10分钟收获细胞。
    11. 倒出介质,将开放的离心管存放在一叠纸巾上2分钟。
    12. 使用真空吸气器清除残留介质的任何液滴 粘附在离心管的壁上或被困在其颈部中。

  2. 冷冻感受态细胞
    1. 将细胞轻轻地悬浮在20ml冰冷的Inoue转化缓冲液中
    2. 加入1.5ml DMSO。 通过涡旋混合细菌悬浮液,然后存储在冰上10分钟
    3. 快速工作,将等分试样的悬浮液分配到冷却的无菌微量离心管中。
    4. 立即通过浸泡紧密冻结感受态细胞   封闭的管在液氮浴中。 将管存储在-70°C 直到需要。 在液氮中冷冻增强转化 效率提高约5倍。 对于大多数克隆目的,50毫升的等分试样 感受态细胞悬浮将是足够的。 但是,当 当需要大量的转化菌落时(例如) 构建cDNA文库),可能需要更大的等分试样。
    5. 需要时,从-70°C冰箱中取出一管感受态细胞。 通过握住手掌中的管解冻细胞。 正如 细胞解冻,将管转移到冰浴中。 将细胞储存在冰上   10分钟。
    6. 使用冷冻,无菌的移液器吸头转移 感受态细胞冷冻,无菌17×100毫米聚丙烯管。 将细胞储存在冰上。 不应使用玻璃管,因为它们较低 转化效率〜10倍。

  3. 转换
    1. 包括所有适当的阳性和阴性对照。
    2. 加入转化DNA(每50 ml感受态细胞最多25 ng)   体积不超过感受态细胞的5%。 旋转管   轻轻地数次混合其内容物。 设置至少两个控制   每个转化实验管,包括一个管子   细菌接受已知量的标准制剂 超螺旋质粒DNA和不接受质粒的细胞管 DNA。 将管在冰上储存30分钟。
    3. 转移管   到放置在预热的42℃循环水浴中的架子。 存储   管在机架正好90秒。 不要摇动管。 热 冲击是关键的一步。 提高细胞是非常重要的 到正确的温度在正确的速率。 孵化 时间和温度已经使用Falcon 2059计算出来了   管。 其他类型的管不一定会产生同等效果 结果。
    4. 快速转移管到冰浴。 让细胞冷却1-2分钟。
    5. 向每个管中加入800ml SOC培养基。 将培养物温热至37℃   水浴,然后将管转移到设置在的摇动培养箱中   37℃。 孵育培养物45分钟以使细菌 恢复并表达由所编码的抗生素抗性标记 质粒。为了最大限度地提高转化效率,轻轻搅拌 (<225周期/分钟)细胞在恢复期间。
    6. 转移适当的体积(高达200毫升/90mm板) 转化的感受态细胞在含有20mM MgSO 4和适当抗生素的琼脂SOB培养基上。选择电阻时 四环素,整个转化混合物可以在a 单板(或在顶层琼脂中电镀)。在这种情况下,收集细菌  通过在微量离心机中在室温(RT)离心20秒,  然后轻轻地将细胞沉淀重悬在100ml的SOC培养基中 轻拍管的侧面。重要通过弯曲的玻璃棒消毒 将其浸入乙醇中,然后在本生灯的火焰中。什么时候 棒已经冷却至室温,将转化的细胞轻轻地铺在其上 琼脂平板的表面。当选择氨苄青霉素抗性时, 转化的细胞应该以低密度(<10 4个 90 mm板),并且板不应孵育超过20小时 在37℃。酶β-内酰胺酶分泌到培养基中 氨苄青霉素抗性转化体,并可快速灭活 抗生素在周围的殖民地。因此, 高密度或孵育它们长时间的结果 出现氨苄青霉素敏感的卫星菌落。 这个问题是 通过使用羧苄青霉素而改善,但不完全消除 而不是在选择性培养基中增加氨苄青霉素 抗生素的浓度从60mg/ml至100mg/ml。 的数量 氨苄青霉素抗性菌落不以线性比例增加 应用于板的细胞数量,也许是因为 生长抑制物质从被杀死的细胞释放 抗生素
    7. 将样品储存在RT,直到液体被吸收
    8. 倒置平板,并在37℃下孵育。 转化的菌落应该出现在12-16小时。

食谱

  1. LB培养基
    每升:向950ml去离子H 2 O中加入
    胰蛋白胨10g
    酵母提取物5 g
    NaCl 10g
    摇动直到溶质溶解。 用5N NaOH(〜0.2ml)将pH调节至7.0。 用去离子H 2 O将溶液的体积调节至1L。 在液体循环中通过在15psi(1.05kg/cm 2 )下高压灭菌20分钟灭菌。
  2. SOB介质
    1. 每升:向950ml去离子H 2 O中加入
      胰蛋白胨20 g
      酵母提取物5 g
      NaCl 0.5g
      摇动直到溶质溶解。 加入10ml的250mM溶液 的KCl(该溶液通过将1.86g KCl溶解在100ml的KCl中而制备 去离子H 2 O)。 用5N NaOH(〜0.2)调节培养基的pH至7.0 ml)。 用去离子H 2 O将溶液的体积调节至1升。 在15psi(1.05kg/cm 2)下在液体上高压灭菌20分钟灭菌 周期。 在使用前,加入5ml的2M MgCl 2的无菌溶液(该溶液通过将19g的MgCl 2溶解在90ml的去离子   H 2 O。 用去离子H 2 O和水调节溶液体积至100ml   通过在15psi(1.05kg/cm 2)下在液体上高压灭菌20分钟来灭菌 循环)。
    2. 含有20mM MgSO 4的SOB琼脂平板和合适的抗生素
    3. 标准SOB含有10mM MgSO 4
    4. SOB培养基,用于培养转化的培养物
    5. 准备三个1升烧瓶250毫升,并平衡培养基到18-20°C,然后接种。
  3. Inoue转换缓冲器
    试剂
    金额/L
    最终浓度
    MnCl 2 4 H sub 2 O
    10.88克
    55 mM
    CaCl 2 2 H sub 2 O
    2.20克
    15 mM
    KCl
    18.65克
    250 mM
    PIPES(0.5M,pH 6.7)
    20ml
    10 mM
    H sub 2 O
    到1 L


    使用前冷却至0℃。
  4. SOC介质
    每个转化反应需要大约1ml的该培养基。 SOC培养基与SOB培养基相同,除了它含有20mM葡萄糖。 在SOB培养基已经高压灭菌后,使其冷却至60℃或更低。 加入20ml无菌1M的葡萄糖溶液(该溶液通过将18g的葡萄糖溶解在90ml的去离子H 2 O中制成。糖溶解后,调节溶液的体积 用去离子H 2 O调节至100ml,并通过0.22μm过滤器灭菌)。
  5. 0.5 M PIPES(pH 6.7)
    将15.1g PIPES溶解在80ml纯H 2 O中

参考文献

  1. Hanahan,D。(1983)。 使用质粒转化大肠杆菌的研究 J Mol Biol em> 166(4):557-580。
  2. Hanahan,D。(1985)。 用于变换的技术。 大肠杆菌。 In DNA cloning:A Practical Approach (ed.D.M.Glover),vol。 1 pp。109-135。 IRL Press,Oxford,United Kingdom。
  3. Inoue,H.,Nojima,H。和Okayama,H。(1990)。 使用质粒高效转化大肠杆菌 < em> Gene 96(1):23-28
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引用:Im, H. (2011). The Inoue Method for Preparation and Transformation of Competent E. coli: "Ultra Competent" Cells. Bio-protocol Bio101: e143. DOI: 10.21769/BioProtoc.143;
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liu chunxia
syngenta
Do you have a try even lower temperature, for example 15°C, 16°C?
6/11/2014 7:07:13 PM Reply
Hogune Im
Molecular and Cellular Pharmacology Program, Department of Pharmacology, University of Wisconsin Medical Schoo, USA

No, I don't think it would make a huge difference but it will grow significantly slower.

6/18/2014 3:32:21 PM