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Gibson Cloning
Author: 杨千惠, updated date: , view: 42, Q&A: 0

Procedure

Gibson Cloning


 

1   试剂的准备

1.1  Phusion DNA Polymerase

货号:NEB#M0530S 2000U/ml  Phusion® Hot Start Flex DNA Polymerase

Catalog #: M0536S , M0536L

 

FunctionHigh Fidelity DNA Polymerases are important for applications in which the DNA sequence needs to be correct after amplification. Phusion High-Fidelity DNA Polymerase offers both high fidelity and robust performance, and thus can be used for all PCR applications. Its unique structure, a novel Pyrococcus-like enzyme fused with a processivity-enhancing domain, increases fidelity and speed. Phusion DNA Polymerase is an ideal choice for cloning and can be used for long or difficult amplicons. With an error rate 50-fold lower than that of Taq DNA Polymerase and 6-fold lower than that of Pyrococcus furiosus DNA Polymerase , Phusion is one of the most accurate thermostable polymerases available. Phusion DNA Polymerase possesses 5´→ 3´ polymerase activity, 3´→ 5´ exonuclease activity and will generate blunt-ended products.

作用:高保真DNA聚合酶在DNA序列扩增后的正确匹配性是重要的。该款DNA聚合酶提供高保真度和执行的随意性,因此可用于所有PCR扩增。根据其独特的蛋白结构,一种新型的似球菌的酶与强化过程的结构域组成,提高保真度和速度。DNA聚合酶是一个理想的克隆选择,可用于长片段或困难片段的扩增。Phusion是最精确的热稳定性聚合酶之一,其错误率比Taq DNA聚合酶低50倍,比糠秕焦球菌DNA聚合酶低6倍。DNA聚合酶具有5’3’聚合酶活性,3’5’外切核酸酶活性,能够产生平末端产物。

1.2  T5 Exonuclease

货号:NEB#M0363S 10000U/ml 

Catalog #: M0363S , M0363L

 

FunctionT5 Exonuclease degrades DNA in the 5´ to 3´ direction . T5 Exonuclease is able to initiate nucleotide removal from the 5´ termini or at gaps and nicks of linear or circular dsDNA . However, the enzyme does not degrade supercoiled ds DNA . T5 Exonuclease also has ssDNA endonuclease activity.

作用:T5核酸外切酶从5’3’降解DNAT5核酸外切酶能够从5’末端或在线性与环状双链DNA的间隙和缺口中开始去除核苷酸。然而,该酶不降解超螺旋的双链DNAT5核酸外切酶也具有单链线性DNA核酸内切酶活性。

 

 

1.3 Taq DNA Ligase

货号:NEB#M0208L 4000U/ml 

Catalog #: M0363S , M0363L

 

FunctionTaq DNA Ligase is a thermostable ligase that catalyzes the formation of a phosphodiester bond between the 5´-phosphate and the 3´-hydroxyl of two adjacent DNA strands. The strands to be ligated need to be hybridized and accurately paired, with no gap, to a complementary DNA strand; allowing resolution of single nucleotide variants. Taq DNA Ligase uses NAD as a cofactor and it is active at elevated temperatures (37° C – 75° C).

作用:Taq DNA Ligase是一种热稳定性连接酶,催化两个相邻DNA链的5-磷酸和3-羟基之间形成磷酸二酯键。要连接的链需要杂交,并且精确配对,没有间隙,与互补的DNA链;允许单核苷酸变异体的解析。Taq DNA连接酶使用NAD作为辅因子,它在升高的温度(37°C75°C)下是活跃的。

 

1.4 NAD

β-Nicotinamide adenine dinucleotide hydrate

 (二磷酸吡啶核苷酸, 辅酶 1, 辅酶 A, 辅酶)

货号:SigmaV900401-5G

Catalog #: V900401 经验分子式(希尔表示法) C21H27N7O14P2 · xH2分子量 663.43 (anhydrous basis) 

 

Function Taq DNA Ligase uses NAD as a cofactor and it is active at elevated temperatures (37° C – 75° C).

作用: Taq DNA连接酶使用NAD作为辅因子,它在升高的温度(37°C75°C)下是活跃的。

 

1.5 PEG 8000

Poly(ethylene glycol)

中文名:聚乙二醇

货号:vetecV900156-500G

Catalog #: V900156线性分子式 H(OCH2CH2)nOH

 

1.6 Tris base

Trizma®

中文名: 2-氨基-2-(羟甲基)-1,3-丙二醇, THAM, Tris , 三羟甲基氨基甲烷, 氨基丁三醇

货号:VetecV900483-500G

Catalog #: V900483线性分子式 NH2C(CH2OH)3  分子量 121.14

 

1.7 Glycerol

中文名: 甘油,丙三醇

货号:国药的10010618

分子式:C2H3O3  分子量 92.09

1.8 EDTA

Ethylenediaminetetraacetic acid

中文名: 乙二胺四乙酸, 亚乙基二次氮基四乙酸, 依地酸

货号:VetecV900106

Catalog #: V900106线性分子式 (HO2CCH2)2NCH2CH2N(CH2CO2H)2  分子量 292.24

 

1.9 NaCl

Sodium chloride

中文名: 氯化钠

货号:VetecV900058

Catalog #: V900058 线性分子式 NaCl  分子量 58.44

 

1.10    DTT

DL-Dithiothreitol

中文名: 二硫苏糖醇

货号:BIOSHARP???

Catalog #:??? 分子式为C4H10O2S2,分子量为154.25

 

1.11    Triton X-100

T-octylphenoxypolyethoxyethanol

中文名: 曲拉通 X-100

货号:生工的A110694-0100

Catalog #: A110694-0100 分子式为C34H62O11,密度 1.06 g/ml(25°C)

 

1.12    MgCl2·6H2O

Magnesium chloride hexahydrate

中文名: 氯化镁 六水合物

货号:VetecV900020

Catalog #: V900020 线性分子式  MgCl2·6H2O  分子量为203.20

 

1.13    dNTP

dNTP mixture, 10 Mm

中文名: dNTP mixture 溶液(10 mM

货号:生工的B500056

Catalog #: B500056

10 mM of each dATP, dCTP, dGTP and dTTP.

 

2   试剂的配置

2.1  1M Tri-Cl pH 7.5

12.114g Tris base 溶于80ml d2H2O,用HCl调节pH到达7.5。补d2H2O定容到到100mL;

 

2.2  50mM NAD

0.03317g NAD(4保存), d2H2O1 ml,配置好后-20℃保存;

 

2.3  5×APB (5×Assembly Pre-Buffer)

母液浓度

所需母液体积

使用浓度

1M Tri-Cl pH 7.5

0.5ml

0.5M Tri-Cl pH 7.5

each dNTP 10mM

100ml

1mM each

50mM NAD

100μl

50mM NAD

PEG8000

250mg

25% m/v

d2H2O

To 1ml

To 1ml

配置好后-20℃保存,使用前用涡旋仪振荡。

 

2.4  1M MgCl2

20.32g MgCl2·6H2O补d2H2O定容到到100mL;

 

2.5  1M NaCl

5.844g NaCl,补d2H2O定容到到100mL

 

2.6  1M DTT

1.5425g DTT补d2H2O定容到到10mL;使用前将沉淀混匀;

 

2.7  0.5M EDTA

14.612 g EDTA,溶于100ml d2H2O,NaOH调节pH到达8。补d2H2O定容到到100mL; 

 

 

 

 

 

 

 

2.8  T5 Exonuclease Dilution Buffer

母液浓度

所需母液体积/μl

使用浓度

100%   glycerol

500

50%   glycerol

1M   Tri-Cl pH 7.5

50

50mM   Tri-Cl pH 7.5

0.5M   EDTA

0.2

0.1mM   EDTA

1M   DTT

1

1mM   DTT

1M   NaCl

100

0.1M   NaCl

Triton   X-100

10

0.1%   Triton X-100 (v/v)

d2H2O

To 1ml

To 1ml

配置好后-20℃保存;

 

2.9  T5(1U/μl)

10μlT5 Exonuclease溶于90μl T5 Exonuclease Dilution Buffer ,配置好后-20保存;

 

2.10          Gibson buffer

母液浓度

所需母液体积/μl

5×APB

26.67

1M   DTT

1.33

1M   MgCl2

1.33

Phusion

1.67

T5(1U/μl)

0.6

Taq   DNA Ligase

13.33

d2H2O

55.07μl (To 100μl)

配置好后分装成1.6μl/管,-20℃保存;其中一次反应只需要1.5μl。

 

 

3   Gibson 反应

3.1   Gibson 反应体系

母液

所需母液体积/μl

片段 (A ng/μl)

x

切好的载体(B ng/μl)

Y

Gibson   buffer

1.5

总体积

2.8


X+Y=1.3

 (A* X)/2= (B* Y)/10

3.2   Gibson 反应程序

50℃,反应45min,反应结束后,直接加入感受态中,或者-20℃保存;

 

 

申明

此Gibson buffer配置方式可与各种试剂官网说明书,一起看。文中方法不一定适合所有的克隆,因此仅供参考。

    如果有错误,请发送到邮箱1329532528@qq.com ,谢谢!


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