Question

consider the bench 4 ligation only, in which the vector is prepared fr directional cloning. how could you do the expereriment if you were given an insert bearing a gene that was back-front, that is the restriction ends on the insert were in the reverse order to the vector-EcoRI/BamHI vs BamHI/EcoRI?

Answer 1

Here, you need to add linkers to the insert with EcoRI and BamHI restriction sites at the each end.

(Linker) EcoR1--------------BamH1------------------------(gene of interest)-------------------------EcoR1 -------------BamH1 (linker)

Now, you need to perform single digestion, for example with EcoR1 that results in the cutting of the interested gene with an extra linker and single restriction site digested vector. When you ligate both, there is a chance of insertion of your interseted in forward direction as per vector restriction sites direction (forward EcoRI/BamHI​). You can confirm it through sequencing.