载体和目的片段连不上，怎么办？ps:Takara T4连接酶..._连接酶_核酸酶类_分子类_知道

游客	收藏 \| 举报 2016-11-17 20:53 关注：241 回答：19 载体和目的片段连不上，怎么办？ps:Takara T4连接酶... 已解决悬赏分：0 - 解决时间 2024-05-08 12:40 载体和目的片段连不上，怎么办？ps:Takara T4连接酶

游客

举报 2016-11-18 16:02

Could you describe your cloning experiments, what insert is, what vector is, how big the insert DNA is, how the insert DNA generated, cut off from other construct, or amplified from DNA or RNA, how the ligation reaction is, what the ligation temperature is, and how long the ligation is incubated? and finally, how is the bacterial transformation done?

游客	举报 2016-11-29 05:01 我们实验室一直在用美国的Clonesmarter 的无缝克隆试剂盒，效果很好，很稳定，您可以试试

游客	举报 2016-11-19 06:36 你回收有问题。小于20ng/ul的回收浓度，一般我都认为是测试误差范围内了，我都认为是不准确的，并且浓度那么低几乎不能用，神马方法都没用。

游客	举报 2016-11-27 00:06 kangaroo0513 你回收有问题。小于20ng/ul的回收浓度，一般我都认为是测试误差范围内了，我都认为是不准确的，并且浓度那么低几乎可是不能用，神马方法都没用。可是为什么我同学回收之后的浓度也是个位数，最后连上了？？？另外，我又重新做了一次，这回浓度高些，但还是没有连上。。。

游客

举报 2016-11-25 22:51

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mpark
Could you describe your cloning experiments, what insert is, what vector is, how big the insert DNA is, how the insert DNA generated, cut off from other construct, or amplified from DNA or RNA, how the ligation reaction is, what the ligation temperature is, and how long the ligation is incubated? and finally, how is the bacterial transformation done?

......

载体9004bp,目的片段1500多bp,按1：3的比例，1:10的也试过，4度过夜和16度过夜都试过，体系20ul和10ul都试过，转化至大肠，就是不长单菌落。阳性对照正常！

游客

举报 2016-11-22 02:22

The vector is over 9 kb, it seems a lentiviral vector. You may need to perform two-step cloning, first try to make subclone of 1.5 kb insert in pUC19 or pBlueScript, or pGEM6 or something like that, or do T-A cloning if the insert is PCR amplified. After you have positive clone, cut off the 1.5 kb insert and try to do ligation with the 9 kb vector. Do transformation using commercial available Stable 2 or Stable 3 competent cells.

游客

举报 2016-11-25 02:37

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mpark
The vector is over 9 kb, it seems a lentiviral vector. You may need to perform two-step cloning, first try to make subclone of 1.5 kb insert in pUC19 or pBlueScript, or pGEM6 or something like that, or do T-A cloning if the insert is PCR amplified. After you have positive clone, cut off the 1.5 kb insert and try to do ligation with the 9 kb vector. Do transformation using commercial available Stable 2 or Stable 3 competent cells.

......

你好，我的载体是酵母表达载体ppic3.5k。。为什么中间要用TA克隆，这样会增加成功率么？？？

游客

举报 2016-11-22 05:00

Nine kb vector may have different genetic elements assembled for multiple function and is different from simple vector for subcloning. It would be quite hard to do direct cloning using this multiple 9 kb vector, specifically for those 1.5 kb or even larger PCR fragment.

Two step cloning will let you run a little bit easy subcloning first and have higher chance to make positive clones. When you have positive subclone, it will facilitate you to go further to have the insert DNA cloned in pPIC3.5K vector.

游客

举报 2016-11-26 19:41

展开引用

mpark
Nine kb vector may have different genetic elements assembled for multiple function and is different from simple vector for subcloning. It would be quite hard to do direct cloning using this multiple 9 kb vector, specifically for those 1.5 kb or even larger PCR fragment.
Two step cloning will let you run a little bit easy subcloning first and have higher chance to make positive clones. When you have positive subclone, it will facilitate you to go further to have the insert DNA cloned in pPIC3.5K vector.

......

你好，，我查了一下资料，有人做构建的时候把目的片段先连接到T载体上，再切下来连接到酵母表达载体上，可是我始终没明白这么做的原理，能否在原理方面解释一下。。请指教！！thank you !