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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.

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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

微生物学 > 微生物细胞生物学 > 细胞分离和培养
作者: Hogune Im
10/20/2011, 9880 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.143

[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.

[Abstract] 本方法与其他实验操作的不同之处在于细菌培养是在18°C 而不是传统的37°C。否则本方法就不值一提,并遵循一个相当标准的过程。为何低温细胞生长会影响转化效率仍是未知。也许18°C合成的细菌膜的成分或物理特性更有利于DNA的摄取,或者有利于高效转化的生长周期阶段延长了。18°C孵育细菌培养基是一个挑战。大多数实验室没有一个在夏季和冬季可以准确地保持温度为18°C的摇床。一种解决方案是将孵化器放置在4℃冷室,并使用温度控制器来加热孵化器至18℃。另外,如果菌液生长在20-23℃几乎没有效率的损失,这是许多实验室的环境温度。在这些温度下菌液孵育培养生长缓慢,倍增时间为2.??5至4h。为了避免后期在夜间达到所需的OD,在晚上建立培养并在第二天早晨早点收菌。这种方法在许多常用分子克隆的大肠杆菌(E. coli)菌株上运作良好 包括XL1-Blue, DH1, JM103, JM108/9, DH5a, 以及 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.

材料与试剂

 

1.         DMSO. DMSO氧化产物转化酶抑制剂, 可能是二甲基,二甲基硫醚(Hanahan 1985). 为了避免出现问题,请购买最高质量的二甲基亚砜。 (Millipore Corp 3p ES-002-10F)

2.         去离子水

3.         酵母提取物

4.         KCl

5.         NaCl

6.         胰化蛋白胨Tryptone

7.         NaOH

8.         MgCl2

9.         MgSO4

10.     抗生素

11.     MnCl2 ·4 H2O

12.     CaCl2 ·2 H2O

13.     PIPES (Alfa Aesar 3p B21835-22)

14.     葡萄糖

 

设备

 

1.        离心机和转子Centrifuges and Rotors (Sorvall GSA 转子或等价物)

2.        Milli-Q 过滤系统(Millipore)

3.        液氮

4.        聚丙烯管(17 x 100 mm; Falcon 2059), chilled in ice

5.        摇床(18°C)

6.        水浴预设为 42°C

7.        Nalgene 过滤器

 

实验步骤

 

在本方法中重要的所有步骤应在无菌条件下进行。

1.        准备细胞

1)         准备Inoue 转化缓冲液(使用前冷却至0°C). 在配制转化缓冲液所用水中的有机污染物可以减少感受态细菌的转化效率。从Milli - Q过滤系统(Millipore)直接获得的水通常能给出好结果。如果问题出现,在使用前用活性炭处理去离子水。

a)         准备0.5 M PIPES (pH 6.7) (piperazine-1,2-bis[2-ethanesulfonic acid],哌嗪-N,N-(2-乙磺酸)) 15.1 g PIPES溶解在80ml纯净水(Milli-Q,或等价物). 5 M KOH 将溶液调至pH 6.7,之后加入纯净水定容至100 ml. 溶液通过一次性预漂洗的Nalgene滤器(0.45μm 孔径)过滤消毒。分成等份,并冷冻储存在-20°C 

b)        准备Inoue转化缓冲液,将后面列出的所有溶质溶解于800 ml纯净水中 ,之后添加20 ml  0.5 M PIPES (pH 6.7). Inoue转化缓冲液定容至1L

c)          通过一个Nalgene 0.45μm的滤器过滤消毒。分成等份,并冷冻储存在-20°C 

2)         从培养皿中挑取37°C培养16-20 h细菌单菌落(直径2-3 mm)。将菌落转接入25 ml LB培养基或SOB培养基的250-ml三角瓶中。在37°C培养6-8 h,剧烈振荡(250-300 rpm)

3)         大约晚上6点,使用此起始培养基转接到31L三角瓶中,每个含有SOB 250 ml。第一个三角瓶转接10 ml,第二个4 ml,第三个2 ml. 18-22过夜培养三个三角瓶,并适度振荡。

4)         第二天早晨,测定三个菌液的OD600值。继续每45 min监测OD值。

5)         当其中一个菌液OD600达到0.55,将培养容器转移到冰水浴10min。弃去其他两个菌液。

6)         大多数实验室环境温度在白天上升并在夜间下降。温度的下降从高峰到低谷的时间变化多少取决于一年中的时间,晚上实验室工作人员数,等等。由于这种变化,很难预测到菌液在任何给定夜晚的生长速率。使用3个不同的培养液以增加过夜培养后获得正确浓度的可能性。

7)         轻轻地在冰冷的80 ml Inoue转化缓冲液中悬浮细胞。细胞最好打重悬而非移液器或涡旋重悬。

8)         4°C10 min离心2500 g (3900 rpm   Sorvall GSA转子) ,收获细胞。

9)         倒掉培养基,并储存在一叠纸巾上的一个开放式离心瓶中2min。使用一个真空吸引器移除残留在离心瓶壁上或是困在其瓶颈部的培养基液滴。

2.        冻结感受态细胞

1)          20 ml 冰冷的Inoue转化缓冲液温和悬浮细胞

2)         加入1.5 ml   DMSO,与细菌悬浮液混合,之后冰上存放10 min

3)         快速操作,将悬浮液等份分装到无菌的离心管中。立即在液氮中速冻密闭管中的感受态细胞。将管储存在-70°C,直到使用。在液氮冷冻可提高转化效率5倍。对于大多数克隆目的,50-ml分装感受态细胞绰绰有余。然而,当需要大量转化克隆时(例如,构建cDNA文库时)需要更大的分装。

4)         需要时将一管感受态从-70°C冰箱取出。用手掌拿管以融化细胞。正当细胞融化时,将管转移到冰浴上。细胞在冰上存放10min

5)         使用一个冷冻的灭菌枪头转移感受态至冷冻的灭菌17 × 100mm的聚丙烯管中。冰上放置细胞。玻璃管不应该使用,因其降低转化效率?10倍。

3.        转化

1)         包括所有适当的阳性和阴性对照。

2)         加入转化DNA(每50-ml感受态细胞可多达25 ng)其体积不超过感受体细胞的5%。轻旋几次管混合。每个转化实验设置至少两个管,包括一管准备的转入已知量标准超螺旋质粒DNA的细菌感受态细胞,以及一管没有任何质粒DNA的细菌感受态细胞。细胞在冰上存放30min

3)         管转移到浮漂上,置于预热42°C循环水浴中,精确90s。不要振荡管。热击非常关键。这里给定的孵育时间和温度,已用Falcon 2059管制定出。其他类型的管不一定会产生相同的结果。

4)         迅速转移管至冰浴上。让细胞冷却1-2 min

5)         每管加入800 ml SOC培养基。将培养基水浴加热到37°C后,转移管到37°C摇床上。 培养菌液45 min使细菌恢复并表达由质粒编码的抗生素抗性标记。为使转化效率最大化,在细胞恢复期间轻轻旋转(<225 /min)

6)        转移适当体积 (200 ml 菌液) 感受体细胞到含有20 mM MgSO4及适宜抗生素的SOB 琼脂培养基中。当选择耐四环素时,整个转化混合物可涂于一个培养基平板(或在琼脂顶层)。在这种情况下,在室温下离心机离心20s收集细菌,之后用100 ml培养基冲到管侧轻轻重悬细胞沉淀。重要的是乙醇浸泡消毒玻璃涂布器后用酒精灯火焰灼烧。当涂布器冷却至室温,将转化细胞温和涂在琼脂培养基表面。当选择氨ampicillin抗性时,转化细胞应在低浓度涂布(每个90-mm 培养基少于<104菌落)。并且培养基不可在37°C培养超过20 h转化的细胞将B-内酰胺酶分泌到培养基中耐氨青霉素,并在菌落周围区域迅速灭活抗生素。因此,涂布高浓度细胞或长时间培养会导致出现氨敏感性的卫星菌落。这个问题得到改善,但还没有完全消除,即在选择性培养基中使用羧青霉素而非氨青霉素,并将抗生素浓度从60 mg/ml提高到 100 mg/ml. 抗性菌落的数量与涂在培养基平板上的细胞不成线形比例增加,也许因为从细胞释放的生长抑制物质被抗生素杀死。

7)         在室温下储存培养基平板,直至液体被吸收

8)         反转板,并在37 °C培养。转化菌落应该在12-16小时出现

 

配方

 

1.         LB (Luria-Bertani)培养基

每升Per Liter: 950毫升去离子水,加入:

蛋白胨 10 g        酵母提取物 5 g       NaCl 10 g

摇动直到溶质溶解。用 5 N NaOH (~0.2 ml)调至pH 7.0 。用去离子水定容至1L. 15 psi (1.05 kg/cm2) 液体循环高压灭菌消毒20 min

2.                     SOB 培养基

1)         每升Per Liter: 950毫升去离子水,加入:

蛋白胨 20 g     酵母提取物 5 g     NaCl 0.5 g

摇动直到溶质溶解。

加入10 ml 250 mM KCl溶液(由溶解在100ml去离子水中的1.86gKCl配制溶液).  5 N NaOH(~0.2 ml)调至pH 7.0。用去离子水定容至1L. 高压灭菌20 min15 psi (1.05 kg/cm2) 液体循环。使用前加入5 ml灭菌的2 M MgCl2溶液。(此溶液由溶解在90ml去离子水中的19 gMgCl2配制。溶液用去离子水定容至100 ml。在15 psi (1.05 kg/cm2) 液体循环高压灭菌消毒20 min) 

2)         SOB琼脂培养基含有20 mM MgSO4及适当抗生素。

3)         标准SOB含有10 mM MgSO4.

4)         SOB培养基用于待转化菌液生长。

5)          准备31升烧瓶(三角瓶)每个250 ml,并在培养前平衡培养基到18-20°C

3.                     Inoue 转化缓冲液

 

试剂

每升所加量

终浓度

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

 

使用前冷却至0℃。

4.                     SOC 培养基

每个转化反应需要这种培养基大约1 ml

SOC 培养基与 SOB培养基是相同的,除了其含有20 mM 葡萄糖。SOB培养基高压灭菌后,让其冷却至60°C或更低。加入20 ml 1 M葡萄糖溶液。 (此溶液由溶解在90ml去离子水中的18 g的葡萄糖配制。溶解后溶液用去离子水定容至100 ml通过一个0.22μm的滤器过滤消毒。

 

参考文献

 

1.        Hanahan D. (1983). Studies on transformation of Escherichia coli with plasmids. Journal of Molecular Biology 166(4): 557-80. 

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., Okayama H. (1990). High efficiency transformation of Escherichia coli with plasmids. Gene 96(1): 23-8. 

 

 

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How to cite this protocol: 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; Full Text



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6/11/2014 7:07:13 PM  

liu chunxia
syngenta

Do you have a try even lower temperature, for example 15°C, 16°C?

6/18/2014 3:32:21 PM  

Hogune Im (Author)
Stanford University

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

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