I want to clone AID enzyme in expression vector so please suggest me which vector will be suitable for this purpose ?.
I am planning to clone AID ( Size approx 4000 bp ) by using PET expression vector. Is this the correct choice for AID gene ?
Plese suggest me the insert capacity size of PET vector?
Hi there,
It is generally accepted that usual bacterial plasmids allow the cloning of insert up to 15kb. In case of pET serie, cloning a 4kb insert shouldn't be a problem. In my lab, the cloning of polycistronic insert encoding for up to 5 different proteins has been succesful.
There are no theoretical limits. It's just that in practice, once you try to clone large (> 10-15 kb) inserts everything becomes harder (ligations are less efficient due to much lower probability of stochastic end-to-end collision, purifications become tricky because of shearing and increased affinity to most common silica resins, transformation is much less efficient, colonies grow much slower due to the increased metabolic burden, etc.) and it becomes preferable to switch to specialized vectors.
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