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This protocol is for making nucleotide changes at specific loci in a large vector (>= 10 kb), and based on QuikChange II XL Site-Directed Mutagenesis Kit (stratagene).

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[Bio101] Site-Directed Mutagenesis
[Bio101] 基因定点突变技术

分子生物学 > DNA > 诱/突变
作者: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
2/5/2011, 10644 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.29

[Abstract] This protocol is for making nucleotide changes at specific loci in a large vector (>= 10 kb), and based on QuikChange II XL Site-Directed Mutagenesis Kit (stratagene).

[Abstract] 这个规程可应用于在一个大载体(>=10kb)上对特异性位点进行核苷酸的改变,同时依据于QuikChange II XL 定点诱变试剂盒 (stratagene)。

Materials and Reagents

  1. QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene)
  2. LB agar (Sigma)
  3. Antibiotics (Sigma)

Equipment

  1. Thermal cyclers (Bio-Rad)

Procedure

  1. Mutant Strand Synthesis Reaction
    1. Prepare the sample reaction as follows:
      5 μl of 10x reaction buffer
      X μl (10 ng) of dsDNA template
      1.25 μl (125 ng) of oligonucleotide primer #1
      1.25 μl (125 ng) of oligonucleotide primer #2
      1 μl of dNTP mix
      3 μl of QuikSolution
      ddH2O to a final volume of 50 μl
      Then add 1 μl of PfuUltra HF DNA polymerase (2.5 U/μl)
    2. Cycle each reaction using the following parameters (be sure to adhere to the 18 cycle limit)

      Segment
      Cycles
      Temperature
      Time
      1
      1
      95 °C
      1 min
      2
      18
      95 °C
      50 sec
      60 °C
      50 sec
      68 °C
      1 min/kb of plasmid length*
      3
      1
      68 °C
      7 min

      *for example, a 5 kb plasmid requires 5 min at 68 °C per cycle
    3. Following cycling, place reaction tubes on ice for 2 min to cool the reactions to 37 °C
    4. Check product by electrophoresis of 10 μl of product on 1% agarose gel. While gel is running, start Dpn1 Digestion
  2. Dpn1 Digestion of the Amplification Products
    1. Add 1 μl of the Dpn1 restriction enzyme (10 U/μl) directly to each amplification reaction using a small, pointed pipet tip
    2. Gently mix each reaction mixture by pipetting up and down several times. Spin for 1 min and incubate the reaction at 37 °C for 1 h to digest the parental supercoiled dsDNA
  3. Transformation of XL10-Gold Ultracompetent Cells
    1. Gently thaw XL10-Gold ultracompetent cells on ice. For each control and sample reaction to be transformed, aliquot 45 μl of the cells to a prechilled polypropylene tube
    2. Add 2 μl of the B-ME mix provided with the kit to the 45 μl cells
    3. Swirl the contents of the tube gently. Incubate on ice for 10 min, swirling gently every 2 min
    4. Transfer 2 μl of the Dpn1-treated DNA from each control and sample reaction to separate aliquots of the ultracompetent cells
      For a positive control, add 1 μl of 0.01 ng/μl pUC18 control plasmid
    5. Preheat NZY + broth in a 42 °C water bath
    6. Heat-pulse tubes in a 42 °C water bath for 30 sec
    7. Incubate the tubes on ice for 2 min
    8. Add 0.5 ml of preheated (42 °C) NZY + broth to each tube, then incubate the tubes at 37 °C for 1 h with shaking 225-250 rpm
    9. Plate the appropriate volume of each transformation rxn as indicated on the table below on proper selection plates

      Reaction Type
      Volume to Plate

      pWhitescript mutagenesis control

      250 μl
      pUC18 transformation control
      5 μl (in 200 μl NZY + broth)
      Sample mutagensis
      250 μl on each of two plates (entire transformation reaction)

    10. Incubate plates at 37 °C for > 16 h

材料和试剂

  1. QuikChange II XL定点诱变试剂盒(Stratagene)
  2. LB琼脂(Sigma)
  3. 抗生素(Sigma)

设备

  1. 热循环仪(Bio-Rad)

程序

  1. 突变链合成反应
    1. 准备样品反应如下:
      5μl10x反应缓冲液
      Xμl(10 ng)的dsDNA模板
      1.25μl(125ng)寡核苷酸引物#1
      1.25μl(125ng)寡核苷酸引物#2
      1μldNTP混合物
      3μlQuikSolution
      ddH 2 O至最终体积为50μl
      然后加入1μl的PfuUltra HF DNA聚合酶(2.5 U /μl)
    2. 使用以下参数循环每个反应(确保遵守18个循环限制)


      周期
      温度
      时间
      1
      1
      95°C /span>
      1分钟
      2
      18
      95°C
      50秒
      60°C
      50秒
      68°C
      1分钟/kb质粒长度*
      3
      1
      68°C
      7分钟

      *例如,5kb质粒在68℃下每个循环需要5分钟
    3. 循环后,将反应管置于冰上2分钟,将反应物冷却至37℃
    4. 通过在1%琼脂糖凝胶上10μl产物的电泳检查产物。 当凝胶运行时,启动Dpn1消化
  2. Dpn1扩增产物的消化
    1. 加入1微升的Dpn1限制酶(10 U /微升)直接到每个扩增反应,使用一个小的尖的吸头尖
    2. 通过上下吹吸几次轻轻混合每个反应混合物。 旋转1分钟,并将反应在37℃下孵育1小时以消化亲本超螺旋dsDNA
  3. XL10-Gold超能量细胞的转化
    1. 轻轻解冻XL10黄金超敏感细胞在冰上。 对于待转化的每个对照和样品反应,将45μl细胞等分至预冷的聚丙烯管
    2. 加入2微升的试剂盒提供的B-ME混合物到45微升的细胞
    3. 轻轻旋转管的内容物。 在冰上孵育10分钟,每2分钟轻轻涡旋
    4. 转移2微升的Dpn1处理的DNA从每个控件和样品反应分开的等分的超敏感性细胞
      对于阳性对照,加入1μl的0.01ng /μlpUC18对照质粒
    5. 在42℃水浴中预热NZY +肉汤
    6. 热脉冲管在42℃水浴中30秒
    7. 孵育管在冰上2分钟
    8. 向每个管中加入0.5ml预热的(42℃)NZY +肉汤,然后在37℃下振荡培养1小时,振荡225-250rpm,
    9. 如下表所示,在适当的选择板
      上铺上适当体积的每个转化rxn
      反应类型
      卷到板

      pWhitescript   mutagenesis control

      250微升
      pUC18转换控制
      5μl(在200μlNZY +培养液中)
      示例 mutagensis
      在两个板(整个转化反应)的每一个上有250μl

    10. 在37℃下孵育平板> 16小时
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How to cite this protocol: Jing, L. (2011). Site-Directed Mutagenesis. Bio-protocol Bio101: e29. DOI: 10.21769/BioProtoc.29; Full Text



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