Dear Abir,

There is no such size limit for cloning. In cloning usually most of the people prefer larger vector and smaller insert e.g. vector of 3kb and insert of 1kb.

In your case your vector is smaller than insert in both cases. You should give a try for cloning it. According to me your vector should behave like insert and vice versa. I feel cloning in case of pGEMT will be easier than cloning in VC155 BiFC. Also, consider ratio of vector:insert and other parameters of ligation reaction such as purity of insert and vector, ligation time and duration.

Best.

Dear Abir

it is certanilly a big fragment and it could be Tricky but not impossibile.

Just some comments:

a problem limitino tour success can be related to the size of you final plasmid (about13kDa) that can be difficult to trasform in standard e

coli strains si you can have that ligation works but your results are negative because your trasformation efficience is very low.

so use a specific strain suitable for big plasmids ( as dh10B or neb-10) or try the PIPE cloning approach which is an enzime free approach where the ligation happen directly inside the cells and thererefore You overcome this problem

You can find information about PIPE cloning on my blog : http://proteocool.blogspot.com/

good luck

Manuele

Yes, it’s possible to clone this size of insert theoritically but very difficult practically. You can do this cloning in two steps. Clone 3-4kb once’s n rest in the same clone .... but take care of the right sequences.

Good luck 👍