NEB/Q5® Site-Directed Mutagenesis Kit (Without Competent Cells)/10 reactions/E0552S,NEB,,NEB,New England Biolabs,纽英伦,+,,Array.Array.Array蚂蚁淘厂家直采.

产品名称：NEB/Q5® Site-Directed Mutagenesis Kit (Without Competent Cells)/10 reactions/E0552S

产品编号：E0552S

市场价：¥0.00

场地：美国(厂家直采)

产品分类：分子类>核酸酶类>其他>

联系QQ：1570468124

电话号码：4000-520-616

邮箱： info@ebiomall.com

美元价：待定

品牌： NEB

公司：New England Biolabs

公司分类：

商品介绍

The Q5 Site-Directed Mutagenesis Kit (Without Competent Cells) enables rapid,site-specific mutagenesis of double-stranded plasmid DNA in less than 2 hours(Figure 1). The kit utilizes the robust Q5 Hot Start High-Fidelity DNA Polymerasealong with custom mutagenic primers to create insertions, deletionsand substitutions in a wide variety of plasmids. After PCR, the amplified materialis added directly to a unique Kinase-Ligase-DpnI (KLD) enzyme mix for rapid(5 minutes), room temperature circularization and template removal (Figure 2).Transformation into high-efficiency chemically-competent E. coli, not supplied,ensures robust results with plasmids up to at least 20 kb in length.Kit is available with competent cells (NEB #E0554)

Figure 1: Site-specific mutagenesis proceeds in less than 2 hours. — The use of a master mix, a unique multi-enzyme KLD enzyme mix, and a fast polymerase ensures that, for mostplasmids, the mutagenesis reaction is complete in less than two hours.

Figure 2: Q5 Site-Directed Mutagenesis Overview. — This kit is designed for rapid and efficient incorporation of insertions, deletions and substitutions into doublestrandedplasmid DNA. The first step is an exponential amplification using standard primers and a master mixfomulation of Q5 Hot Start High-Fidelity DNA Polymerase. The second step involves incubation with a uniqueenzyme mix containing a kinase, a ligase and DpnI. Together, these enzymes allow for rapid circularization of thePCR product and removal of the template DNA. The last step is a high-efficiency transformation into chemicallycompetentcells (not provided).

Figure 3: Primer Design for Q5 Site-Directed Mutagenesis — Substitutions, deletions and insertions are incorporated into plasmid DNA through the use of specifically designedforward (black) and reverse (red) primers. Unlike kits that rely on linear amplification, primers designedfor the Q5 Site-Directed Mutagenesis Kit should not overlap to ensure that the benefits ofexponential amplification are realized. A) Substitutions are created by incorporating the desired nucleotidechange(s) (denoted by *) in the center of the forward primer, including at least 10 complementary nucleotides onthe 3´side of the mutation(s). The reverse primer is designed so that the 5´ends of the two primers anneal backto-back. B) Deletions are engineered by designing standard, non-mutagenic forward and reverse primers that flankthe region to be deleted. C) Insertions less than or equal to 6 nucleotides are incorporated into the 5´ end of theforward primer while the reverse primer anneals back-to-back with the 5´ end of the complementary region of theforward primer. D) Larger insertions can be created by incorporating half of the desired insertion into the 5´ endsof both primers. The maximum size of the insertion is largely dictated by oligonucleotide synthesis limitations.

Q5 Graph — Results from a substitution reaction (4 nt) using the back-to-back Control SDM Primer Mix and Control SDM Plasmid (6.7 kb) are shown, along with results from a 12 nt deletion experiment (5.8 kb plasmid) and an 18 nt insertion experiment (7.0 kb plasmid). In all three cases, over 90% of the resultant colonies had incorporated the desired mutation(s). Results are normalized to total transformants if cells were not diluted prior to plating. For comparison, the same substitution reaction (4 nt) was performed with the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent) following Agilent’s protocol and using Agilent’s primer design tool to design overlapping primers.*Note that the QuikChange kit does not accommodate deletions and insertions of this size, so no comparison could be made for these experiments.

This product is related to the following categories:: DNA Assembly, Cloning and Mutagenesis Kits Products

This product can be used in the following applications:: Site Directed Mutagenesis,; Site Directed Mutagenesis

品牌介绍

NEB公司成立于二十世纪七十年代中期，拥有众多经验丰富的科学家，是生产生命科学试剂的领导者。目前，NEB为基因组研究提供最齐全的重组酶和天然酶，并且公司业务范围已延伸至蛋白质组学和药品开发领域。回顾三十余年来的历程，NEB公司作为先驱公司之一，为促进生物科技工业的发展做出了巨大的贡献。NEB美国总部乔迁新址后拥有最尖端的设备，有一座现代化的发酵中心及设备齐全的实验室，这些实验室主要用于产品生产、质量监控、产品开发和基础科研之用。作为首批以商业规模生产限制性内切酶的公司之一，NEB一直专注于内切酶的研究，并保持业内领先水平。NEB公司一贯坚持以科学为本的原则，公司生产的试剂因其高质量、高性价比享誉世界。NEB（北京）有限公司 New England Biolabs (Beijing) LTD. NEB（北京）有限公司New England Biolabs (Beijing) LTD. 为美国New England Biolabs, Inc.在华投资兴建的独资子公司，负责处理其在中国内地及香港地区的全部业务，同时承担部分新产品的研发工作。纽英伦生物技术（北京）有限公司成立于2001年11月，为中国生命科学研究者提供一流的产品和专业的技术服务，促进国内外生命科学学术交流，与中国科研工作者一起将中国推入到二十一世纪生命科学的前沿中去。

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