I am supposed to construct a plasmid that contains features from two other plasmids. My strategy is to generate three fragments form the two other plasmids. I was encouraged to try Gibson assembly, so I went to NeBuilder entered my sequences and got I list of primers with overhangs and ordered them… But now I am I bit unsure about the linearization of the backbone and insert into that fragment.
In NeBuilder you can choose "by sequence position" in for the insert in the backbone the description reads “Entering the sequence positions of the bases immediately flanking the insert site. Primers for inverse PCR terminating at these endpoints will be calculated automatically”.
Does anyone have experience using NeBuilder? i am completely unfamiliar with inverse PCR ? also my regular software package Geneious is unable to comprehend the two primers for the inverse PCR reaction