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K123240 Thermo PCR克隆,CloneJET™ PCR Cloning Kit with DH10B Competent Cells/含DN10B感受态细胞的PCR克隆试剂盒

2024-10-24

CloneJET™ PCR Cloning Kit with DH10B Competent Cells/含DN10B感受态细胞的PCR克隆试剂盒

货号：K123240,K123120

规格：40 reactions,20 reactions

价格：4458,2362

产品类型：PCR 及DNA聚合酶

品牌：Thermo Fisher

This package includes the CloneJET PCR Cloning Kit (20 or 40 reactions) and DH10B Competent Cells that are optimized for use with the kit and can be directly transformed with the ligation product.CloneJET PCR Cloning KitThe Thermo Scientific CloneJET PCR Cloning Kit is an advanced positive-selection system for high-efficiency cloning of PCR products generated with any thermostable DNA polymerase. Any blunt or sticky-end DNA fragment can be cloned. The kit works well with both phosphorylated and non-phosphorylated DNA fragments. Ligation into the included positive-selection vector takes only five minutes, yielding more than 99% recombinant clones. Blunt-ended PCR products generated with a proofreading enzyme are ligated directly into the cloning vector.PCR products generated either with non-proofreading DNA polymerases or mixtures of DNA polymerases need to be blunted prior to ligation in a five minute reaction with the thermostable DNA Blunting Enzyme provided with the kit. DH10B Competent Cells are optimized for use with the CloneJET PCR Cloning Kit and can be directly transformed with the ligation product.The CloneJET PCR Cloning Kit contains the novel, ready-to-use positive selection cloning vector pJET1.2/blunt. The vector contains a lethal restriction enzyme gene that is disrupted by ligation of a DNA insert into the cloning site. As a result, only bacterial cells with recombinant plasmids are able to form colonies. Re-circularized pJET1.2/blunt vector molecules lacking an insert express a lethal restriction enzyme, which kills the host E. coli cell after transformation. This positive selection drastically accelerates the process of colony screening and eliminates additional costs required for blue/white selection.DH10B Competent CellsThermo Scientific DH10B Competent Cells are high efficiency, chemically competent E.coli cells. The DH10B strain is suitable for cloning DNA containing methylcytosine and methyladenine, allowing both prokaryotic and eukaryotic genomic DNA to be cloned efficiently. These cells are ideal for the construction of cDNA libraries using plasmid-derived vectors. Genotype: F– mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ (ara-leu)7697 galU galK λ– rpsL(StrR) nupG.• High transformation efficiency: >1x109 cfu/µg pUC19 DNA• Suitable for routine and high-throughput cloning applications• Convenient two reactions per vial packaging• S.O.C. Outgrowth medium included

优点：

- 快捷——可在短短5分钟内克隆 PCR 产物- 多功能性——适用于磷酸化或非磷酸化的DNA 片段，以及平末端和粘性末端 PCR 产物- 高效——可获得超过99%的阳性重组克隆，且无克隆背景- 经济——无需昂贵的蓝白斑筛选- 兼容性强——可直接转化进常用E. coli菌株，包括 TOP10 和 XL1-Blue

数据：

图1.DH10B competent cell transformation efficiency in CloneJET workflowA 976-bp PCR product was generated using either Thermo Scientific DreamTaq Green PCR Master Mix (2X) (Cat. No. K1081, represented by grey bar) or Thermo Scientific Phusion Hot Start II DNA Polymerase (Cat. No. F549, represented by blue bar) and ligated into a cloning vector according to the manufacturers’ protocols. The transformation efficiency obtained with the control fragment provided with the CloneJET PCR Cloning Kit is represented by the yellow bar. Ligation mixtures were used to transform Thermo Scientific DH10B cells. The expected transformation efficiency of competent cells is >1 x 107CFU/µg DNA. The transformation efficiency specification of DH10B Competent Cells with pUC19 DNA is 1 x 109CFU/µg DNA.

组成成分：

▪20 x DH10B Competent Cells for Subcloning (100 µL)▪ 2 x pUC19 DNA (20 µL) (100 pg/µL)▪ 2 x S.O.C. Medium (5 mL)▪ pJET1.2/blunt Cloning Vector (46 µL) (50 ng/µL)▪ 2X Reaction Buffer (600 µL)▪ T4 DNA Ligase (46 µL) (5 U/µL)▪ DNA Blunting Enzyme (46 µL)▪ DNA Blunting Enzyme (46 µL)▪ pJET1.2 Forward Sequencing Primer, 10 µM aqueous solution (50 µL)▪ pJET1.2 Reverse Sequencing Primer, 10 µM aqueous solution (50 µL)▪ Control PCR Product, 976 bp with 3’-dA overhangs (8 µL) (12 ng/µL)▪ Water, nuclease-free, (1.25 mL)

K123240 Thermo PCR克隆,CloneJET™ PCR Cloning Kit with DH10B Competent Cells/含DN10B感受态细胞的PCR克隆试剂盒

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