n*y
2 楼
现在有一个质粒， 里面已经有一个gene A 了， 然后需要在A的中间插入一段序列，
有800 bp， 没有合适的 restrction sites 可以用， 有什么办法吗？多谢
有800 bp， 没有合适的 restrction sites 可以用， 有什么办法吗？多谢
c*y
3 楼
不懂，用EAD怎么跳？
【在 y*****n 的大作中提到】
: 用ead跳槽就是爽！！！
【在 y*****n 的大作中提到】
: 用ead跳槽就是爽！！！
d*p
4 楼
You can PCR the Gene A fragment before the insertion sit(A), after the
insertion site(B), as well as the 800bp(C). Then using fusion PCR to stick
them together. There should be at least 30bp overlaping region between A and
C; B and C. maybe there is another more easy way.
【在 n****y 的大作中提到】
: 现在有一个质粒， 里面已经有一个gene A 了， 然后需要在A的中间插入一段序列，
: 有800 bp， 没有合适的 restrction sites 可以用， 有什么办法吗？多谢
insertion site(B), as well as the 800bp(C). Then using fusion PCR to stick
them together. There should be at least 30bp overlaping region between A and
C; B and C. maybe there is another more easy way.
【在 n****y 的大作中提到】
: 现在有一个质粒， 里面已经有一个gene A 了， 然后需要在A的中间插入一段序列，
: 有800 bp， 没有合适的 restrction sites 可以用， 有什么办法吗？多谢
X*n
5 楼
楼上正解，google:
OE-PCR
CPEC cloning
OE-PCR
CPEC cloning
n*y
6 楼
多谢， 但是A很大， 分成两部分之后， 分别是1kb 和5kb， overlap pcr可行吗？
and
【在 d**p 的大作中提到】
: You can PCR the Gene A fragment before the insertion sit(A), after the
: insertion site(B), as well as the 800bp(C). Then using fusion PCR to stick
: them together. There should be at least 30bp overlaping region between A and
: C; B and C. maybe there is another more easy way.
and
【在 d**p 的大作中提到】
: You can PCR the Gene A fragment before the insertion sit(A), after the
: insertion site(B), as well as the 800bp(C). Then using fusion PCR to stick
: them together. There should be at least 30bp overlaping region between A and
: C; B and C. maybe there is another more easy way.
j*n
7 楼
可以用Gibson assembly
或者homologous recombination based cloning
★ 发自iPhone App: ChineseWeb 7.8
【在 n****y 的大作中提到】
: 多谢， 但是A很大， 分成两部分之后， 分别是1kb 和5kb， overlap pcr可行吗？
:
: and
或者homologous recombination based cloning
★ 发自iPhone App: ChineseWeb 7.8
【在 n****y 的大作中提到】
: 多谢， 但是A很大， 分成两部分之后， 分别是1kb 和5kb， overlap pcr可行吗？
:
: and
n*y
8 楼
多谢， 第二个是在 yeast 里面做的？
【在 j****n 的大作中提到】
: 可以用Gibson assembly
: 或者homologous recombination based cloning
:
: ★ 发自iPhone App: ChineseWeb 7.8
【在 j****n 的大作中提到】
: 可以用Gibson assembly
: 或者homologous recombination based cloning
:
: ★ 发自iPhone App: ChineseWeb 7.8
d*p
9 楼
Can you find the restriction enzyme site less than 2kb distance from the
insertion site? If yes, you do not have amplify very long fragments. Similar
size of fragments can joint together easier than different size fragments.
Another way: You can amplify the whole plasmid( How big is the plasmid?) by
the primers at the insertion site. Then you can introduce the enzyme site in
the primer. After PCR amplify the plasmid, digest it and ligate your 800bp
fragment and transform. You need to amplify your 800bp fragment with the
same Enzyme site in the primer.
【在 n****y 的大作中提到】
: 多谢， 但是A很大， 分成两部分之后， 分别是1kb 和5kb， overlap pcr可行吗？
:
: and
insertion site? If yes, you do not have amplify very long fragments. Similar
size of fragments can joint together easier than different size fragments.
Another way: You can amplify the whole plasmid( How big is the plasmid?) by
the primers at the insertion site. Then you can introduce the enzyme site in
the primer. After PCR amplify the plasmid, digest it and ligate your 800bp
fragment and transform. You need to amplify your 800bp fragment with the
same Enzyme site in the primer.
【在 n****y 的大作中提到】
: 多谢， 但是A很大， 分成两部分之后， 分别是1kb 和5kb， overlap pcr可行吗？
:
: and
l*y
10 楼
red/et recombination system in e.coli
【在 n****y 的大作中提到】
: 多谢， 第二个是在 yeast 里面做的？
【在 n****y 的大作中提到】
: 多谢， 第二个是在 yeast 里面做的？
N*n
11 楼
Does anyone have the Red/ET e.coli strain? I need to my some plasmids but it
doesn't make sense to license it only for some subcloning. I can pay you.
Please PM.
doesn't make sense to license it only for some subcloning. I can pay you.
Please PM.
z*h
12 楼
Simple Cloning: direct transformation of PCR product (DNA multimer) to
Escherichia coli and Bacillus subtilis
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.
http://aem.asm.org/content/early/2011/12/16/AEM.07105-11.abstra
Escherichia coli and Bacillus subtilis
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.
http://aem.asm.org/content/early/2011/12/16/AEM.07105-11.abstra
H*i
13 楼
LS的不就是CPEC么 和09年写的http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006441有什么不同？
哦 有点不同，多PCR了几个循环，产物弥散状
反正我两片段PCR连接 P出弥散的或者正好质粒大小都挺常见的。
哦 有点不同，多PCR了几个循环，产物弥散状
反正我两片段PCR连接 P出弥散的或者正好质粒大小都挺常见的。
N*n
14 楼
Actually, there is a plasmid that can do the same job of recombineering for
plasmid, BAC or genomic DNA recombination. Just bought it.
plasmid, BAC or genomic DNA recombination. Just bought it.
b*n
15 楼
不用全PCR出来
5kb中找出一个合适的酶切位点，离你要插入位点几百到2kb之内都行
P好之后酶切连回同样酶切的原模板质粒上
【在 n****y 的大作中提到】
: 多谢， 但是A很大， 分成两部分之后， 分别是1kb 和5kb， overlap pcr可行吗？
:
: and
5kb中找出一个合适的酶切位点，离你要插入位点几百到2kb之内都行
P好之后酶切连回同样酶切的原模板质粒上
【在 n****y 的大作中提到】
: 多谢， 但是A很大， 分成两部分之后， 分别是1kb 和5kb， overlap pcr可行吗？
:
: and
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