m*********r
发帖数: 98 | 1 有时你需要克隆一个片断，用两个不同的酶切位点， Ｘ and Y. 但是它们在载体上相
距很近， 比如说只有几个碱基。在切载体时，这带来两个问题： １是不好切，比如说
某个酶需要保护碱基；２ 是不容易判断消化是否完全，因为单切和双切后的载体在胶
上看上去完全一样。
如果你需要经常用到这个载体， 或者having a hard time cloning something into
site X and Y, 那就值得花点时间做这个：随便克隆一个dummy片断 （１kb左右），
用X 位点单切。 克隆到载体上的X点后，就可以作为你今后的出发载体了。今后要克隆
东西到 X+Y点时， 先用 Y 切，检查线性化完全后， 再用 X 切， 释放你原先放上的
dummy. 胶检，回收，就可以放心用了。
Of cause, another way is to ask around to see if anybody has already cloned
something into the X+Y sites in this vector, if you are lucky to find one,
u | t****t
发帖数: 610 | 2 CIP can solve the problem.
Case 1: no cut with either enzyme. The supercoil can be seperated by gel if
you run it long and slow enough.
Case 2: single cut. Although it is almost identical to the double cut
plasmid, it cannot be ligated to the insert, which has two different sticky
ends. The only possibility is self ligation. CIP can prevent or make it less
possible.
However, if you use blunt ends, that's another story and I agree with OP. |
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