Team:Tuebingen/Results
Mutagenisation of the RFC10 restriction sites and the ApaI restriction site in the backbone of the pRS plasmids were performed by mutagenesis PCR. Positive clones were screened by restriction analysis with the respective restriction enzymes (compare plasmid maps: link [project description pRS vectors]).
Then, the old multiple cloning site (MCS) was replaced by restriction digest (ApaI, SacI) and subsequent ligation of annealed oligonucleotides containing matching overhangs. Verification of successful replacement of the old MCS was performed by using a restriction site that was only present in the initial pRS MCS (SalI).
Figure 1: Schematic agarose gel showing the migration pattern of various pRS plasmids incubated with different restriction enzymes.
Figure 2: Restriction analysis of various pRS plasmid constructs.
In figure 1, a schematic agarose gel showing the migration pattern of the three pRS vectors when digested with different restriction enzymes is depicted. Figure 2 shows the restriction analysis of various pRS plasmid constructs. The used pRS vectors show the expected migration behaviour. Lane 3 in figure 2c shows an additional fragment at around 5 kb which indicates an incomplete digestion of the pRS316 plasmid. In summary, every RFC10 restriction site in the pRS plasmid backbones was removed and the MCS was replaced. Unfortunately, due to time constraints we were not able to insert a terminator.