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- 询价记录
- 文献和实验
- 技术资料
- 保存条件:
冰袋(wet ice)运输。产品-20℃保存。有效期1年。
- 保质期:
有效期1年
- 供应商:
翌圣生物
- 库存:
大量
- 英文名:
Hieff Mut® Site-Directed Mutagenesis Kit 定点突变试剂盒
- 规格:
10 T(11003ES10)
产品描述:
Hieff Mut™ Site-Directed Mutagenesis Kit是基于Hieff Clone™快速克隆技术的定点突变系统。使用本试剂盒,目标质粒扩增产物经DpnI消化、Hieff Clone™重组环化后直接进行转化即可完成定点突变。该试剂盒由两个模块组成:Hieff® II Pfu DNA Polymerase扩增模块和Hieff Clone®快速克隆模块。Hieff® II Pfu DNA Polymerase超高的保真度显著降低了扩增过程中引入新突变的可能性,其卓越的长片段扩增能力,广泛适用于长度小于20 kb的任何质粒扩增。Hieff Clone®快速克隆系统利用高效的同源重组反应替代传统的退火成环反应。因此使用Hieff Mut™ Site-Directed Mutagenesis Kit进行DNA定点突变时,引物设计更加灵活,且扩增反应不再需要以线性方式进行,极大减少了模板使用量,有利于原始甲基化模板的彻底降解。Hieff Clone®技术可以高效完成两个PCR产物的无缝拼接,因此该试剂盒还能以单次扩增的方式对目标质粒上不连续的两个位点同时进行突变。
Hieff Mut™ Site-Directed Mutagenesis Kit中的重组酶Exnase经过优化,专门针对单碱基、不连续双碱基定点突变。此外,如扩增产物特异,其DpnI消化产物可不进行DNA纯化而直接用于重组反应。高度优化的反应缓冲液、快捷的操作流程以及极高的成功率,使得Hieff Mut™ Site-Directed Mutagenesis Kit成为DNA定点突变试剂盒。
产品属性:
| 热启动 | 否‑ |
| 退火温度 | 60-72℃‑ |
| 保真度 | 83倍‑ |
| 产品形式 | Kit‑ |
| 应用 | DNA定点突变、单点突变‑ |
| 延伸时间 | 30-60s/kb‑ |
| 反应速度 | 20min‑ |
| 电泳指示剂 | 否‑ |
产品特色:
- 专门针对单碱基、不连续双碱基定点突变
应用案例:
- 单碱基(或连续多碱基)定点突变实验方案

- 不连续双碱基定点突变实验方案 (两突变位点相距超过50 bp)

储存条件:
冰袋(wet ice)运输。产品-20℃保存。有效期1年。
常见问题:
Q:定点突变克隆原理是什么?
A:基于同源重组技术的定点突变系统。使用本试剂盒,目标质粒扩增产物经 DpnI 消化 同源重组环化后直接进行转化即可完成定点突变。
Q:多点突变试剂盒与定点突变试剂盒有什么区别?
多点突变试剂盒可一步实现目标质粒上 3-5 个不连续位点的定点突变,而定点突变试剂盒是适合 1 或2 个不连续位点的定点突变。如:3 个连续的碱基突变算 1 个位点的突变,故定点突变试剂盒即可(相距超过 50 bp 为 1 个不连续位点)。
Q:点突变为什么要选择甲基化的载体?
A:点突变过程需要去除模板质粒,防止假阳性。利用的原理就是限制性内切酶 DpnI 能消化甲基化的模板,若是非甲基化,则无法去除模板。
Q:质粒反向扩增时引物如何设计?
A:正反向扩增引物 5’端包含至少 20 bp 反向互补区域,各引物非互补区域长度至少为 20 bp。互补区尽量选择无重复序列,GC 含量尽量在 40%-60%范围内。
Q:质粒模板的使用量?
A:在不影响扩增产物的情况下,尽可能少的量(建议<1ng),避免DpnI 消化不完全。
Q:DpnI 处理后要进行回收吗?
A:较优进行胶回收。未消化完全的质粒或其他非特异性条带会影响后续的重组反应。
Q:对于PCR 模板质粒什么要求?
A:PCR 模板应为甲基化质粒,应使用甲基化酶无缺陷型的宿主菌扩增原始质粒(如DH5α,TOP10, JM109 等)。应尽量使用新鲜制备的质粒作为模板,因长期放置,反复冻融可能会导致模板质粒断裂,开环或降解。
Q:适合多大的质粒模板进行突变?
A:小于 20 kb。质粒越大,其他非目的碱基的突变率越高,PCR 更难,重组效率更低。
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Multiple Site-Directed Mutagenesis In Vitro
Polymerase chain reaction (PCR)-based site-directed mutagenesis (SDM) methods are widely used in molecular biology to selectively alter gene sequences (1 ). However, common protocols allow for the introduction
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Site-directed mutagenesis techniques are invaluable tools in molecular biology to study the structural and functional properties of a protein. To expedite the time required and simplify methods for mutagenesis, we recommend
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