Direct Transfer Cloning
The Direct Transfer Cloning procedure is recommended in cases the optimal expression and purification system is already known. In this procedure, a PCR product containing the gene of interest can be directly inserted into the appropriate Acceptor Vector without the need for prior generation of a Donor Vector.
In a first step the PCR fragment is generated using the respective forward (CF) and reverse (CR) primer extending the gene of interest (GOI) with the corresponding integration sites. This PCR product is in the second step integrated into the appropriate Acceptor Vector resulting in the final expression construct, the so called Destination Vector. The formation of the correct Destination Vector is monitored via blue-white screening on LB-Agar plates containing X-Gal. The validated Destination Vector can be directly used for transformation or transfection of the expression host.