Hello. According to your experiences.. So far, it was not possible to successfully link my insert-vector. Vector: I gave him a treatment with alkaline phosphatase and then washed with phenol/ chloroform. Subsequently ligased 16 hours at 16 °C, however in this step it happens that my vector does not bind with my insert but the same. What new protocol do you recommend?
By stating that the vector was still the same, do you mean that you have no colonies after plating or you have colonies but without the insert?
Maybe you can try to change the molar ratio, let's say you are ligating one insert and one vector, you can increase the molar ratio to vector:insert = 1:5 to increase the chances of ligation.
Which method are you using for ligation? I'll recommend the Hot-Fusion method: Article Hot Fusion: An Efficient Method to Clone Multiple DNA Fragme...
It worked as well as Gibson assembly if not better.
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