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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, 10219 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

材料与方法

1.     QuikChange II XL Site-Directed Mutagenesis Kit (stratagene)

2.     LB 琼脂 (sigma)

3.     抗生素 (sigma)

 

设备

1.     Thermal cyclers (Bio-rad)

 

步骤

1.     突变位点合成反应

1)     准备如下反应试剂

10X 反应缓冲液   5ul

单链DNA模板     10ng

引物1            1.25ul (125ng)

引物2            1.25ul (125ng)

dNTP                      1ul

3ul  QuikSolution

ddH20  调节终体积到50ul

1ul PfuUltra HF DNA 聚合酶 (2.5U/ul)

2)     按以下步骤循环反应 (至少18个循环)

步骤

循环数

温度

时间

1

1

95

1 minute

2

18

95

50 seconds

60

50 seconds

68

1 minute/kb of plasmid length*

3

1

68

7 minutes

*, 5kb 的质粒需要第二个步骤的68 反应时间为 5 分钟

3)     循环后将反应的管置冰上2分钟,使反应物温度降到37

4)     1%琼脂糖凝胶电泳检测产物,上样量10 ul,电泳同时开始Dpn1处理。

2.     扩增产物的Dpn1处理

1)     每个扩增产物管加 1ul Dpn1内切酶(10U/ul)

2)     轻轻混匀每个反应管,离心, 37 孵育1小时消化单链模板DNA

3.     XL10-Gold Ultracompetent 细胞转化

1)     轻轻将 XL10-Gold ultracompetent 细胞放冰上.每个样品或对照取45 ul 细胞到预冷离心管。

2)     每个离心管加2ul B-ME 与细胞混合

3)     轻轻混匀离心管,冰浴10分钟,每隔2分钟轻微混匀

4)     Dpn1处理后的DNA加入离心管中,每管2ul。正对照:加 1ul 0.01ng/ul pUC18 质粒

5)     42度水浴预热NZY+培养基

6)     离心管在42度水浴热激30

7)     迅速将离心管置冰上,2分钟

8)     每管加0.5ml预热的NZY+培养基,将离心管置37度摇1小时,转速225-250 rpm

9)     每管取适当体积的转化产物涂抗生素筛选培养板

反应类型

涂板体积

P诱变对照

250ul

pUC18 质粒

5ul (in 200ul NZY+ broth)

样品诱变基因

250ul on each of two plates (entire transformation reaction)

10)  将板放置37 培养16小时以上

English
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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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