r******y
发帖数: 21907 | 1 先是克隆到pENTR里面，有两个觉得对 可是测序后发现给插反了，是不是还得重新来过
，这样克隆到destination里面就不成了吧？或者说根本无法克隆到destination
vertor里面？我的片断3.5 kb。前几天的2 kb的没反差问题。。。 | c**g
发帖数: 14 | 2 1. use pENTR/D vector, it has direction preference.
2. wrong direction can still be cloned into destination vector, however,
your gene of interest would be expressed 3' to 5'. | z*h
发帖数: 773 | 3 try this new technology -- Simple Cloning.
You, C., X.-Z. Zhang, and Y.-H. P. Zhang. 2012. Simple Cloning: direct
transformation of PCR product (DNA multimer) to Escherichia coli and
Bacillus subtilis. Appl. Environ. Microbiol.:doi:10.1128/AEM.07105-07111.
Abstract
We developed a general restriction enzyme-free and ligase-free method for
subcloning up to three DNA fragments into any location of a plasmid. The DNA
multimer generated by prolonged overlap extension PCR was directly
transformed in Escherichia coli [e.g., TOP10, DH5α, JM109, BL21(DE3)] and
Bacillus subtilis for obtaining chimeric plasmids. | g*********5
发帖数: 2533 | 4 would you pls give me a copy? uc system can't open it.
pls mail to g*******[email protected], thx a lot.
DNA
【在 z*h 的大作中提到】
: try this new technology -- Simple Cloning.
: You, C., X.-Z. Zhang, and Y.-H. P. Zhang. 2012. Simple Cloning: direct
: transformation of PCR product (DNA multimer) to Escherichia coli and
: Bacillus subtilis. Appl. Environ. Microbiol.:doi:10.1128/AEM.07105-07111.
: Abstract
: We developed a general restriction enzyme-free and ligase-free method for
: subcloning up to three DNA fragments into any location of a plasmid. The DNA
: multimer generated by prolonged overlap extension PCR was directly
: transformed in Escherichia coli [e.g., TOP10, DH5α, JM109, BL21(DE3)] and
: Bacillus subtilis for obtaining chimeric plasmids.
| s******s
发帖数: 13035 | 5 谁有这个paper？我连不上
不过好像是VT的，不敢学啊
DNA
【在 z*h 的大作中提到】
: try this new technology -- Simple Cloning.
: You, C., X.-Z. Zhang, and Y.-H. P. Zhang. 2012. Simple Cloning: direct
: transformation of PCR product (DNA multimer) to Escherichia coli and
: Bacillus subtilis. Appl. Environ. Microbiol.:doi:10.1128/AEM.07105-07111.
: Abstract
: We developed a general restriction enzyme-free and ligase-free method for
: subcloning up to three DNA fragments into any location of a plasmid. The DNA
: multimer generated by prolonged overlap extension PCR was directly
: transformed in Escherichia coli [e.g., TOP10, DH5α, JM109, BL21(DE3)] and
: Bacillus subtilis for obtaining chimeric plasmids.
| s******a
发帖数: 472 | 6 VT 是什么？
【在 s******s 的大作中提到】
: 谁有这个paper？我连不上
: 不过好像是VT的，不敢学啊
:
: DNA
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