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 p... 阅读全帖 |
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g*********3
发帖数: 177 | 2 感谢大家的帮助～
前天看到有同学回贴，谈到在5-3LTR之间插入Poly A的问题，有篇文章http://www.ncbi.nlm.nih.gov/pubmed/18627247 有一些结论
BTW，大家能否给些建议关于weak promoter，在lentivirus中的prmoter很多都太强，
如果我想要subendogenous的蛋白表达量，哪种promoter比较好用～
老板推荐说pBABE/pLXSH的LTR promoter不错，不过我查了下文献 好像证据不是很多。
做过的同学给点建议～
先谢过大家了 |
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g*********3
发帖数: 177 | 3 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. |
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g*********3
发帖数: 177 | 4 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. |
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L*****t
发帖数: 56 | 5 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。 |
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