x********u
发帖数: 430 | 1 Is it possible that I insert a 6Kb fragment into a 8Kb vector? I have
repeated three times and have no luck geting any colonies. For construct
below 10Kb, I am very confident (>80%) and I can always get the right
transformants after screening. Is there any tricks for this big fragment
cloning? I know all my gene can be functionally expressed in E coli, so
there is no protein toxicity issue. | h**********r
发帖数: 671 | 2 应该问题不大吧，我做过6 kb以上的。载体也正好是8 kb左右。感觉AT克隆比直接单酶
切连接载体（去磷酸化）效率还要低。
要多挑克隆倒是真的。 | s******s
发帖数: 13035 | 3 多挑克隆就行了？切好纯化好，colony pcr筛个100个
【在 x********u 的大作中提到】
: Is it possible that I insert a 6Kb fragment into a 8Kb vector? I have
: repeated three times and have no luck geting any colonies. For construct
: below 10Kb, I am very confident (>80%) and I can always get the right
: transformants after screening. Is there any tricks for this big fragment
: cloning? I know all my gene can be functionally expressed in E coli, so
: there is no protein toxicity issue.
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