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北京石油化工学院2026年研究生招生接收调剂公告
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applexixixi

木虫 (小有名气)


[交流] 想扩增10kb多的基因簇,现在有什么技术可以一次性扩增出来吗

10kb多的基因簇是连续的,想扩增出来,在此问问虫友们现在有什么技术可以解决这个问题呢?
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huiee

金虫 (正式写手)


★ ★ ★
applexixixi(金币+1): 谢谢参与
小丹木木: 金币+2, 鼓励交流 2012-10-27 13:46:40
推荐一篇paper 如果愿意用的话
Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting

    Jun Fu,       
    Xiaoying Bian,       
    Shengbaio Hu,       
    Hailong Wang,       
    Fan Huang,       
    Philipp M Seibert,       
    Alberto Plaza,       
    Liqiu Xia,       
    Rolf Müller,       
    A Francis Stewart       
    & Youming Zhang       

    Affiliations
    Contributions
    Corresponding authors

    Nature Biotechnology
    30,
    440–446
    (2012)
    doi:10.1038/nbt.2183

Received
    24 October 2011
Accepted
    16 March 2012
Published online
    29 April 2012

Functional analysis of genome sequences requires methods for cloning DNA of interest. However, existing methods, such as library cloning and screening, are too demanding or inefficient for high-throughput application to the wealth of genomic data being delivered by massively parallel sequencing. Here we describe direct DNA cloning based on the discovery that the full-length Rac prophage protein RecE and its partner RecT mediate highly efficient linear-linear homologous recombination mechanistically distinct from conventional recombineering mediated by Redαβ from lambda phage or truncated versions of RecET. We directly cloned all ten megasynthetase gene clusters (each 10–52 kb in length) from Photorhabdus luminescens into expression vectors and expressed two of them in a heterologous host to identify the metabolites luminmycin A and luminmide A/B. We also directly cloned cDNAs and exactly defined segments from bacterial artificial chromosomes. Direct cloning with full-length RecE expands the DNA engineering toolbox and will facilitate bioprospecting for natural products.
18楼2012-10-26 16:51:13
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weigh1111

铁杆木虫 (正式写手)



applexixixi(金币+1): 谢谢参与
你这够呛吧……分两三段用高保真酶PCR出来再连接呗
2楼2012-10-24 19:17:23
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polepolar

银虫 (初入文坛)



applexixixi(金币+1): 谢谢参与
可以试一下Phusion聚合酶,据说可以一次扩增10k多.
3楼2012-10-24 21:43:12
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wuyun123

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applexixixi(金币+1): 谢谢参与
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8楼2012-10-24 21:55:46
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