Efficient Subcloning using the StarGate System
Fig. 1:
Using StarCombinaseTM the gene for GFP was transferred from a Donor Vector into a pPSG-IBA Acceptor Vector. After an 1 hour incubation, E. coli was transformed with the reaction mixture. All resulting clones exhibited green fluorescence by GFP expression. This provides evidence that the StarGate subcloning reaction had performed accurately and reliably to put the GFP gene under control of the T7 promoter.
Reaction Efficiency >99%
Fig. 2:
Transformation of E. coli with a StarGate transfer reaction mixture typically leads to more than thousand white colonies harbouring the desired Destination Vector. The presence of a few blue colonies only indicates a reaction efficiency far beyond 99%.
Transfer Efficiency is Independent of Gene Size
Fig. 3 and 4:
The green fluorescent protein (GFP, 700 bp), bacterial alkaline phosphatase (BAP, 1400 bp) and T7 RNA polymerase (T7RNAP, 2650 bp) were transferred from the respective Donor Vector into all available Acceptor Vectors (cf. Table, page 4). The result provides evidence that the StarGate subcloning procedure is almost not influenced by the length of the transferred gene and that even large genes can be transferred efficiently.
|
