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进口试剂

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CopyControl™ HTP Fosmid Library Production Kit

进口试剂

CopyControl™ HTP Fosmid Library Production Kit

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产品名称:

CopyControl™ HTP Fosmid Library Production Kit

货号
CCFOS059
规格
1 Kit
存储温度
MaxPlax is stored at -70℃, the remainder is stored at -20℃
没有此类产品
产品描述

由于生产线调整,自2017年1月1日起,此款产品转至Lucigen™品牌销售, (仅品牌变化,产品外包装变化,本身没有改变,老客户可以继续使用,实验不受影响)

 

Epicentre FOSMID文库构建试剂盒

 

应用

可完成各种生物样本编制完整和无偏向的Fosmid文库,利用自动诱导液保证单拷贝和诱导高拷贝数量的需要。
可以用水和土壤等含有微生物的环境样品构建宏基因组文库。

CopyControl™Fosmid文库构建试剂盒提供了高效改良的建库方法,可以构建约40 kb克隆的文库。
CopyControl pCC1FOS™载体包含大肠杆菌F因子单拷贝复制起点以及可诱导高拷贝oriV。
CopyControl Fosmid克隆通常培养于单拷贝中,以确保毒蛋白和不稳定DNA测序的插入的定性和成功克隆。
在DNA纯化前Fosmid克隆可以快速诱导达到每个细胞50拷贝。这一步,大大增加了DNA产量,同时保持质粒的稳定。
CopyControl HTP Fosmid文库包含pCC2FOS载体,它是针对末端测序优化设计的,尤其在高通量测序方面。
与Agencourt生物科技公司联合设计的引物,消除了昂贵的不必要的引物测序……
此外,在每个引物3'末端的七个碱基序列是专门设计,以尽量减少对模板纯化后大肠杆菌DNA的任何误引导。
试剂盒通过随机剪切DNA,采用克隆平端DNA片段的策略,以产生更加完整和公正的,可以通过部分限制性内切酶消化获得的基因库。
首先将基因组DNA剪切成约40 kb的片段。剪切的DNA片段经过末端修复变成钝末端,然后经过5'-磷酸化、大小筛选,然后从低融点琼脂糖凝胶回收。最后,将大小选定的DNA接到cloning-ready CopyControl pCC1FOS 载体或者 pCC2FOS载体上。用试剂盒附带的高效MaxPlax™拉姆达包装蛋白(包装效率>109 pfu/µg)进行包装,最后接种到附带的EPI300细胞中。

优点

•CopyControl pCC1FOS和pCC2FOS载体:线性化,去磷酸化,纯化,已经为连接做好准备。
•不需要限制性内切酶消化或脉冲凝胶电泳来准备用于克隆的基因组DNA。
•使用pCC2FOS载体可以最大化高通量末端测序结果。
•可以诱导单拷贝到每个细胞50拷贝。安全地获得较高的DNA产量,同时保持了稳定克隆单拷贝数。
•高效率的lambda包装蛋白消除了背景值假阳性。
•比BAC克隆更快更容易。

Applications

  • Preparation of complete and unbiased fosmid libraries, from any biological sample, that can be maintained at single-copy number and induced to high-copy number as needed, using the CopyControl™ Autoinduction Solution.
  • Construction of metagenomic libraries from microbes present in environmental samples such as water and soil.

The CopyControl™ Fosmid Library Production Kits* provide an efficient and improved method for constructing a library of approximately 40 kb clones. The CopyControl pCC1FOS™ Vector contains both the E. coli F-factor single-copy origin of replication and the inducible high-copy oriV (Fig. 1). CopyControl Fosmid clones are typically grown at single copy to ensure insert stability and successful cloning of encoded and expressed toxic protein and unstable DNA sequences. The CopyControl Fosmid clones can then be induced up to 50 copies per cell immediately before DNA purification. This step greatly increases DNA yields, while maintaining the stability of the plasmid.

The CopyControl HTP Fosmid Library contains the pCC2FOS™ Vector which is designed to optimize end-sequencing results, especially in a high-throughput setting. The primer cassette, engineered in conjunction with Agencourt Bioscience Corporation, eliminates wasteful and expensive vector sequence reads by having the 3´ ends of the primer-annealing sites only three bases from the vector/insert junction. In addition, the seven-base sequence at the 3´ end of each primer was specifically designed to minimize mispriming to any contaminating E. coli DNA present after template purification (Fig. 2).

The kit uses a strategy of cloning blunt-ended DNA fragments generated by random shearing of the DNA, to produce more complete and unbiased genomic libraries than can be obtained by partial restriction endonuclease digests. Genomic DNA is first sheared into approximately 40-kb fragments. The sheared DNA is end-repaired to generate blunt, 5´-phosphorylated ends and then size-selected by and recovered from a low-melting-point agarose gel. Finally, the size-selected DNA is ligated into the cloning-ready CopyControl pCC1FOS or pCC2FOS Vector, packaged using ultra-high efficiency MaxPlax™ Lambda Packaging Extracts (>109 pfu/µg DNA), included in the kit, and plated on the supplied TransforMax™ EPI300™ E. coli (Fig. 3).

Figure 1. CopyControl™ Vector map. The CopyControl pCC1FOS™ and pCC2FOS™ Vectors for CopyControl Fosmid library production are supplied linearized at the Eco72 I (blunt) site and then dephosphorylated. The vector is ready for cloning end-repaired (blunt-end) genomic DNA of approximately 40 kb.

Figure 2. The CopyControl™ pCC2FOS™ Vector primer cassette. The vector differs from the pCC1FOS Vector by the engineering of a new primer cassette that eliminates wasteful vector-derived sequencing reads and minimizes the potential for priming on the E. coli genome.

Benefits

  • CopyControl pCC1FOS and pCC2FOS Vectors are supplied Cloning-Ready: linearized, dephosphorylated, purified, and ready for ligation.
  • No need for partial restriction endonuclease digests or pulse field gel electrophoresis to prepare the genomic DNA for cloning.
  • Maximize high-throughput end-sequence results using the pCC2FOS Vector (Fig. 4).
  • Clones can be induced from single copy up to 50 copies per cell (Fig. 5). Safely obtain higher DNA yields while maintaining the stability of single-copy-number clones.
  • High-efficiency lambda packaging eliminates background and false positives.
  • Faster and easier than BAC cloning.

Figure 3 (click to enlarge). Overview of the process for preparing a fosmid library using the CopyControl™ Fosmid Library Production Kits. Once the library has been prepared, individual clones can be cultured in small volume and induced to multiple-copy number for high yields of high-purity DNA for fingerprinting, sequencing, etc., using EPICENTRE's DirectLysis Fosmid96 kit or FosmidMAX™ DNA Purification Kit.

Figure 4. Typical sequencing results obtained with the pCC2FOS™ Forward Primer on a pCC2FOS™ clone at 1/48x BigDye™ dilution. Similar results were obtained with the pCC2FOS Reverse Primer (data not shown).

Citations

  1. FEMS Microbiol Lett 284 (2008) 28-34

*Covered by issued and/or pending patents.

 

Figure 5. CopyControl™ Fosmid clones can be induced up to 50 copies per cell to greatly increase DNA yield. Hind III digests of fosmid DNA isolated from uninduced (–) and induced (+) CopyControl clones. Digests contained one-third (8 µl) of the total sample volume and were analyzed by agarose gel electrophoresis. Lane M, Kilobase ladder.

 

 

 Catalog No. Concentration Size

CopyControl™ Fosmid Library Production Kit
   CCFOS110   1 Kit
For producing up to 10 complete and unbiased fosmid libraries.

Contents: CopyControl™ pCC1FOS™ Fosmid Vector, End-Repair Enzyme Mix, End-Repair 10X Buffer, dNTP Mix, Fast-Link™ DNA Ligase, Fast-Link™ 10X Ligation Buffer, ATP Solution, GELase™ Enzyme Preparation, GELase™ 50X Reaction Buffer, MaxPlax™ Lambda Packaging Extracts, Fosmid Control DNA, Ligated Lambda Control DNA, EPI300™ Plating Strain, Control Lambda Plating Strain, CopyControl™ Fosmid Autoinduction Solution.


CopyControl™ HTP Fosmid Library Production Kit
   CCFOS059   1 Kit
For producing up to 10 complete and unbiased fosmid libraries.

Contents: CopyControl™ pCC2FOS™ Fosmid Vector, End-Repair Enzyme Mix, End-Repair 10X Buffer, dNTP Mix, Fast-Link™ DNA Ligase, Fast-Link™ 10X Ligation Buffer, ATP Solution, GELase™ Enzyme Preparation, GELase™ 50X Reaction Buffer, MaxPlax™ Lambda Packaging Extracts, Fosmid Control DNA, Ligated Lambda Control DNA, EPI300™ Plating Strain, Control Lambda Plating Strain, CopyControl™ Fosmid Autoinduction Solution.


pCC1/pEpiFOS-5 Forward Sequencing Primer
   F5FP010 50 µM 1 nmole

pCC1/pEpiFOS-5 Reverse Sequencing Primer
   F5RP011 50 µM 1 nmole

pCC2 Forward Sequencing Primer
   HTFP061 50 µM 1 nmole

pCC2 Reverse Sequencing Primer
   HTRP062 50 µM 1 nmole

CopyControl™ Induction Solution
   CCIS125   25 ml
1,000X concentrated solution. Filter-sterilized.

CopyControl™ Fosmid Autoinduction Solution
   A1S107F   50 ml

 

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