StarGate® Entry Cloning via pENTRY-IBA
Step 1: Entry Cloning for Donor Vector generation
In the first step, the gene of
interest (GOI) will be equipped at both termini with combinatorial sites and
the LguI recognition sites, which are
important for oriented insertion of the PCR fragment into pENTRY-IBA51. This is
done by PCR using a proofreading polymerase.
Recombination of the PCR product with the Entry Vector at the combinatorial sites (red and orange) leads to generation of the Donor Vector. This step involves loss of the LguI restriction sites (dark orange with arrowheads), making the recombination reaction unidirectional and thereby highly efficient.
In the resulting Donor Vector the same combinatorial sites are now flanked by the Esp3I recognition sites (light blue), thereby enabling a highly efficient and specific StarGate® gene transfer process into Acceptor Vectors in a similar manner (link: Step2).