s*r
2 楼
在做一个targeting vector，总长17kb多。现在做到最后一步克隆了，把一个4.4kb的
片段用BamHI切下来，装到BclI线性化的12kb backbone里。已经反复做了几次，每次8-
12miniprep里面90%都是一个大小3kb的奇怪质粒,偶尔出来的几个大小正确的测序发现
全部都是反向。先前怀疑是由于质粒太大在E.coli里发生重组，这次换成了stbl3的感
受态，还是一样的结果。求有经验的给点建议...
片段用BamHI切下来，装到BclI线性化的12kb backbone里。已经反复做了几次，每次8-
12miniprep里面90%都是一个大小3kb的奇怪质粒,偶尔出来的几个大小正确的测序发现
全部都是反向。先前怀疑是由于质粒太大在E.coli里发生重组，这次换成了stbl3的感
受态，还是一样的结果。求有经验的给点建议...
w*d
3 楼
I encountered a similar problem previously.
Someone told me that writhing of a circular DNA may occur when the insertion
is too long. If this cannot be tolerated during replication, the circular
DNA will not be amplified.
Finally, I changed another vector to fix it.
Someone told me that writhing of a circular DNA may occur when the insertion
is too long. If this cannot be tolerated during replication, the circular
DNA will not be amplified.
Finally, I changed another vector to fix it.
m*5
4 楼
by which method did you dephorsphorylate your plasmid? I usually got most of
plasmid self ligased
plasmid self ligased
c*g
5 楼
CIP from NEB is pretty good.
of
【在 m******5 的大作中提到】
: by which method did you dephorsphorylate your plasmid? I usually got most of
: plasmid self ligased
of
【在 m******5 的大作中提到】
: by which method did you dephorsphorylate your plasmid? I usually got most of
: plasmid self ligased
s*r
6 楼
Yes, I CIPed the vector for 1h, but it didnt appears to be self ligation.
The 3kb plasmid is weird, because the vector itself is 12kb.
The 3kb plasmid is weird, because the vector itself is 12kb.
a*p
7 楼
17k还用酶切，还是单酶切？ 真牛呀。 用重组吧
m*5
8 楼
??? detail??
【在 a******p 的大作中提到】
: 17k还用酶切，还是单酶切？ 真牛呀。 用重组吧
【在 a******p 的大作中提到】
: 17k还用酶切，还是单酶切？ 真牛呀。 用重组吧
a*p
9 楼
A highly efficient recombineering-based method for generating conditional
knockout mutations
看看这片文章，然后朝作者要个能重组的菌
knockout mutations
看看这片文章，然后朝作者要个能重组的菌
I*a
10 楼
让E。coli长慢一点，
以前遇到这种情况，
我每次都筛选几十个或是上百个克隆，有时候会有对的，
以前遇到这种情况，
我每次都筛选几十个或是上百个克隆，有时候会有对的，
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