g*********3
发帖数: 177 | 1 大家好。
最近一段时间在挣扎gateway LR reaction:
用的是pLXSH gateway 作为destination ector
用的另一个entry ector含有attL1 ATTL2
所用的质粒测序无误含有recombination site。
用的是invitrogen的LR cronase enzyme mix II.
pLXSH is resistant to Amp and After LR reaction, I do transformation. I didn
't get any colony. [Zero.]
Entry vector is amp resistant too. So I cut the amp out and linearize this
vector.
I also do LR reaction with plxsh + Gus. Gus is positive control entry vector
provided by the kit.
There is no colony.
I have done positive pUC19 for transformation. it works.
I called the company and they say LR enzyme mix is very sensitive and I need
avoiding freeze-thaw.
Actually I use the new kit and it doesn't work.
各位给点建议，看有可能哪些步骤出问题了。实在觉得可疑。
另外我的DNA is dissoved in TE and the LR reaction is 8ul (DNA and fill it
into 8ul with TE) + 2ul enzyme mix.
Does TE matters? This is the only difference between my recipe and my
labmate. Her LR works.
谢谢啦。 |
g*********3
发帖数: 177 | 2
didn
【在 g*********3 的大作中提到】
: 大家好。
: 最近一段时间在挣扎gateway LR reaction:
: 用的是pLXSH gateway 作为destination ector
: 用的另一个entry ector含有attL1 ATTL2
: 所用的质粒测序无误含有recombination site。
: 用的是invitrogen的LR cronase enzyme mix II.
: pLXSH is resistant to Amp and After LR reaction, I do transformation. I didn
: 't get any colony. [Zero.]
: Entry vector is amp resistant too. So I cut the amp out and linearize this
: vector.
|
g*********3
发帖数: 177 | 3
didn
【在 g*********3 的大作中提到】
: 大家好。
: 最近一段时间在挣扎gateway LR reaction:
: 用的是pLXSH gateway 作为destination ector
: 用的另一个entry ector含有attL1 ATTL2
: 所用的质粒测序无误含有recombination site。
: 用的是invitrogen的LR cronase enzyme mix II.
: pLXSH is resistant to Amp and After LR reaction, I do transformation. I didn
: 't get any colony. [Zero.]
: Entry vector is amp resistant too. So I cut the amp out and linearize this
: vector.
|
X******n
发帖数: 914 | 4 你不会用了LB＋cm＋ AMP的plate了吧？ |
g*********3
发帖数: 177 | 5 When I prepared PLXSH-gateway, I use Amp+Chloramphenicol.
After LR, I use AMp only to do transformation and miniprep.
[At this point, CCDB is out and I don't use Chloramphenicol.]
Is this what you mean?
Thanks a lot.
【在 X******n 的大作中提到】
: 你不会用了LB＋cm＋ AMP的plate了吧？
|
g*********3
发帖数: 177 | 6 问题可能有点简单 让大伙见笑了啊。。。。
【在 X******n 的大作中提到】
: 你不会用了LB＋cm＋ AMP的plate了吧？
|
s******s
发帖数: 13035 | 7 应该用TE的，这个没问题。
LR的酶爆烂，容易死。
didn
【在 g*********3 的大作中提到】
: 大家好。
: 最近一段时间在挣扎gateway LR reaction:
: 用的是pLXSH gateway 作为destination ector
: 用的另一个entry ector含有attL1 ATTL2
: 所用的质粒测序无误含有recombination site。
: 用的是invitrogen的LR cronase enzyme mix II.
: pLXSH is resistant to Amp and After LR reaction, I do transformation. I didn
: 't get any colony. [Zero.]
: Entry vector is amp resistant too. So I cut the amp out and linearize this
: vector.
|
b******s
发帖数: 1089 | 8 LR reaction sometimes gives odd outcome, such as no colonies or false
colonies. But mostly it should be very reliable.
You can try to gel purify both entry clone and destination clone and do LR
reaction immediately.
didn
【在 g*********3 的大作中提到】
: 大家好。
: 最近一段时间在挣扎gateway LR reaction:
: 用的是pLXSH gateway 作为destination ector
: 用的另一个entry ector含有attL1 ATTL2
: 所用的质粒测序无误含有recombination site。
: 用的是invitrogen的LR cronase enzyme mix II.
: pLXSH is resistant to Amp and After LR reaction, I do transformation. I didn
: 't get any colony. [Zero.]
: Entry vector is amp resistant too. So I cut the amp out and linearize this
: vector.
|
g*********3
发帖数: 177 | 9 Yes. It is highly possible that my DNA quality is bad.
So I redo the midiprep of plxsh this week and do digestion to run gel---
check quality.
It is good.
The insert may be bad because it is digested and gel purified.
But I don't understand why the Gus positive doesn't give any result.
Thanks for your advice.
【在 b******s 的大作中提到】
: LR reaction sometimes gives odd outcome, such as no colonies or false
: colonies. But mostly it should be very reliable.
: You can try to gel purify both entry clone and destination clone and do LR
: reaction immediately.
:
: didn
|
z*t
发帖数: 863 | 10 我之前也有过同样的问题。。。不过我使用invitrogen pDonor vector作为donor
我后来用了1uL的recominase,四度过夜后室温3小时，PK 37C 30min(好麻烦-_-||)
后面colony的postive rate能达到90%左右
还有mannual上说supercoiled plasimid间的recombination效率最高，试一试用非AMP
抗性的supercoiled vector吧
didn
【在 g*********3 的大作中提到】
: 大家好。
: 最近一段时间在挣扎gateway LR reaction:
: 用的是pLXSH gateway 作为destination ector
: 用的另一个entry ector含有attL1 ATTL2
: 所用的质粒测序无误含有recombination site。
: 用的是invitrogen的LR cronase enzyme mix II.
: pLXSH is resistant to Amp and After LR reaction, I do transformation. I didn
: 't get any colony. [Zero.]
: Entry vector is amp resistant too. So I cut the amp out and linearize this
: vector.
|
|
|
a***o
发帖数: 129 | 11 why don't you use entry plasmids with kanR? Is it likely when you cut out
the ampR there is a hidden restriction site between attL1 and L2 and you don
't know about? |
g*********3
发帖数: 177 | 12
AMP
【在 z*t 的大作中提到】
: 我之前也有过同样的问题。。。不过我使用invitrogen pDonor vector作为donor
: 我后来用了1uL的recominase,四度过夜后室温3小时，PK 37C 30min(好麻烦-_-||)
: 后面colony的postive rate能达到90%左右
: 还有mannual上说supercoiled plasimid间的recombination效率最高，试一试用非AMP
: 抗性的supercoiled vector吧
:
: didn
|
g*********3
发帖数: 177 | 13 Yes. It is better to use different resistant genes. I don't plan well enough
at the beginning.
I check the digestion map and XMNI/BSAI is unique to cut out part of Amp
fragment.
Thanks anyway.
don
【在 a***o 的大作中提到】
: why don't you use entry plasmids with kanR? Is it likely when you cut out
: the ampR there is a hidden restriction site between attL1 and L2 and you don
: 't know about?
|
L*****t
发帖数: 56 | 14 你把pLXSH空载体转DB3.1之类耐ccdB的菌株，Amp+CM平板划线挑几个克隆做Miniprep再
试试看。
我以前听人提过慢病毒系列的Gateway载体因为重复序列较多，存放久了构象会改变导
致克隆失败，但是本身序列没变所以重新转化提新鲜的质粒就行了。 |
g*********3
发帖数: 177 | 15 谢谢大家的建议。LR终于work了。
尼玛的没别的，就是LR enzyme mix不稳定，重新买了新的， 就工作了。
买了three boxes of enzyme mix. only one of them works!
发誓再也不用这货了，非常不稳定，后来用那个work的enzyme (stored at -20)做了第
二遍，就不work了。
无语了。 |
s******s
发帖数: 13035 | 16 哈哈，我当天就说是酶的问题，给我乌鸦嘴说中了。
不过这新酶就死，我还没遇见过，你可以打电话要求退钱。基本上
这玩意适合一次克隆N个的实验室，开了就用完。如果每次用个1ul,
三五次以后我看就没戏了。
Btw, 我们实验室都是阴谋论，大家讨论了一下，极端怀疑那个酶
里面掺了点蛋白酶。
【在 g*********3 的大作中提到】
: 谢谢大家的建议。LR终于work了。
: 尼玛的没别的，就是LR enzyme mix不稳定，重新买了新的， 就工作了。
: 买了three boxes of enzyme mix. only one of them works!
: 发誓再也不用这货了，非常不稳定，后来用那个work的enzyme (stored at -20)做了第
: 二遍，就不work了。
: 无语了。
|
s********n
发帖数: 2939 | 17 还好吧，我之前用的那管还是别人两年前用剩下的，我还克隆了好几个，都是100%没问
题。 |
