With a Not I site at each end, the NRG insert of SK13b1 can be ligated into the vector in two directions. However, the NRG gene can only be expressed when the insert is oriented in the same direction as the cmv promoter. The orientation of the insert can be tested by digesting the plasmid with an enzyme that cuts the plasmid once inside the insert and once in the vector. When the insert is oriented correctly, digesting with Eco RV yields bands that are 2504 and 5937 base pairs. When the insert is oriented backwards, digesting with Eco RV produces bands that are 826 and 7615 base pairs.
Gel electrophoresis is used to determine the size of the digested pieces.
The samples were run uncut, after digestion with Eco RV to test the direction of the insert, and after digestion with Not I to ensure that the Not I sites were recreated during ligation. A 1 Kb ladder and lambda-Hind III ladder were run as standards. All of the clones shown above contain the NRG insert in the desired orientation. CHO cells were transfected with pcDNA6v5lacZ, a version of the vector used in SK13b1 construction that also contains a b-galactosidase gene. The b-galactosidase gene causes cells to turn blue when treated with a staining solution.