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NGS Library Prep kits

Molecular Research

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ClonExpress II One Step Cloning Kit

ClonExpress® technology is a simple, fast and efficient DNA seamless cloning technology, which can clone the insert into any position of any vector. Linearize the vector and place the insert 5'in the forward/reverse PCR primer Introduce the end sequence of the linearized vector into the end, so that the 5'and 3'ends of the PCR product have the same sequence as the two ends of the linearized vector (15 bp-20 bp). This PCR product and the linearized vector are in a certain ratio After mixing, under the catalysis of the recombinase, the reaction can be carried out at 37°C for 30 min to complete the directional cloning. As a new generation recombinant cloning kit, ClonExpress® II has a unique non-ligase dependent system, which greatly reduces the background of vector self-ligation, and the positive rate can reach more than 95%. The highly optimized reaction buffer and the enhanced recombinase Exnase II significantly improve the recombination efficiency of cloning and the tolerance to impurities, making it possible for linearized vectors and inserts to be directly used for recombination cloning without purification, greatly simplifying The experimental steps.    Simple, fast, efficient, suitable for any carrier; No need to consider the restriction site carried by the insert; Efficient cloning of 50 bp-10 kb DNA fragments; Linearized cloning vector and PCR products can be cloned directly without purification; No ligase, no self-connection, positive rate >95%   Store at  -20°C
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Number of views:
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Product serial number
C112-01/02
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ClonExpress® technology is a simple, fast and efficient DNA seamless cloning technology, which can clone the insert into any position of any vector. Linearize the vector and place the insert 5'in the forward/reverse PCR primer Introduce the end sequence of the linearized vector into the end, so that the 5'and 3'ends of the PCR product have the same sequence as the two ends of the linearized vector (15 bp-20 bp). This PCR product and the linearized vector are in a certain ratio After mixing, under the catalysis of the recombinase, the reaction can be carried out at 37°C for 30 min to complete the directional cloning.
As a new generation recombinant cloning kit, ClonExpress® II has a unique non-ligase dependent system, which greatly reduces the background of vector self-ligation, and the positive rate can reach more than 95%. The highly optimized reaction buffer and the enhanced recombinase Exnase II significantly improve the recombination efficiency of cloning and the tolerance to impurities, making it possible for linearized vectors and inserts to be directly used for recombination cloning without purification, greatly simplifying The experimental steps. 

 

Simple, fast, efficient, suitable for any carrier;
No need to consider the restriction site carried by the insert;
Efficient cloning of 50 bp-10 kb DNA fragments;
Linearized cloning vector and PCR products can be cloned directly without purification;
No ligase, no self-connection, positive rate >95%

 

Store at  -20°C

Keyword:
C112-01/02
We could not find any corresponding parameters, please add them to the properties table

Q1:引物如何设计?

A1:(1) 引物设计:官网引物设计软件CE Design V1.04,选择相应模块进行设计;

(2) 载体的线性化方式有三种:双酶切、单酶切和反向PCR,优先选择双酶切;

(3) 引物由三部分组成:同源臂(15-20 bp,不计算酶切位点和残留碱基,GC含量40%-60%)+酶切位点(根据实验需求保留或舍弃)+特异性引物(引物Tm值的计算不包括同源臂的序列)。

Q2:平板上长不出克隆或者克隆数很少。

A2:(1) 引物设计不正确:引物包含15-20 bp同源臂(不计算酶切位点),GC含量40%-60%;若同源臂的GC含量过高或过低,可重新选择插入位点;

(2) 线性化克隆载体和插入片段扩增产物的使用量不足/过量,即比例不佳:尽量按照说明书推荐的量和比例配置重组体系;

(3) 重组体系不低于10 μl,各组分添加不低于1μl(避免低体积添加造成量取不准);若载体或片段浓度过低导致添加量超过20 μl,可同比例降低载体和片段的添加量;插入片段长于载体时,重组体系中片段与载体的摩尔比为2:1;

(4) 载体和插入片段不纯,抑制反应:未纯化DNA不应超过4 μl(反应体系体积的1/5);建议线性化载体和PCR产物进行胶回收纯化,纯化产物溶解在pH 8.0的ddH2O保存;

(5) 感受态细胞的转化效率需大于107 cfu/μg,重组产物的转化体积不应超过感受态细胞体积的1/10,否则会降低转化效率;选择克隆用感受态细胞(如DH5α/XL10),不能选择表达用感受态细胞;

(6) 酶切获得的载体应经加热处理失活限制性内切酶,对于加热处理不能失活的酶切体系,必须经过胶回收纯化;

(7) 核查基因是否致死。

Q3:多数克隆含有不正确的插入片段。

A3:PCR产物混有非特异性产物:优化PCR体系,提高特异性;胶回收PCR产物;鉴定更多的克隆。

Q4:插入片段出现碱基突变。

A4:插入片段在PCR获得时使用高保真的聚合酶;若突变发生在同源臂区域,请进行引物序列核对。

Q5:多数克隆不含插入片段。

A5:(1) 克隆载体线性化不完全:可通过阴性对照检测载体是否线性化完全;对于优化酶切体系,提高限制性内切酶使用量、延长酶切反应时间、胶回收纯化酶切产物;

(2) 插入片段获得的PCR体系中混入了相同抗性的质粒:PCR扩增模板为环状质粒时,如扩增产物直接用于重组反应时需进行DpnI消化,或者进行胶回收纯化。

Q6:阳性对照不长斑或很少。

A6:(1) 平板抗性使用错误:试剂盒中提供的对照载体的抗性为A+抗性;

(2) 感受态细胞效率低:确保转化效率>107cfu/μg,可进行简单检测,将1 ng质粒转化至100 μl感受态细胞中,取1/10进行涂板,生长1000个菌斑,估算感受态转化效率为107cfu/μg;重组产物的转化体积不应超过感受态细胞体积的1/10,否则会降低转化效率;感受态选择克隆感受态(如DH5α/XL10),不能用表达感受态。

Q7:菌液PCR无条带。

A7:(1) 引物不正确:建议使用载体的通用引物进行菌检,或至少使用一条通用引物;

(2) PCR体系或程序不合适:没有目的条带也没有空质粒条带,建议优化PCR体系、程序;或者提取质粒,以质粒做模板PCR验证,或着进行酶切验证;

(3) 重组失败:只有空质粒的条带,说明重组不成功,载体线性化不完全,建议优化酶切体系。

Q8:菌种(表达用)保存一段时间后出现表达量下降。

A8:菌种在保存过程中,可能会出现质粒丢失,拷贝数降低的情况,重新培养时适当增加抗生素的浓度,筛选出含高拷贝数质粒的细菌。

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