Team:Bilkent UNAM Turkey/Experiment

We ordered our genes. We optimized codon usage because of gene taken from Enterobacter cloacae.

Codon Usage Then we did restriction digestion and gel electrophoresis.
We repeated this step a lot because of failuire.I put right result

We used standard gel purification kit Our ligation style is really similar to iGEM's one.
Our one;

insert volume(µl):X

Cut Vector Volume(µl):X

DEPC-water(µl):8,5-2X

T4 DNA ligase(µl):0,5

Ligation Buffer(µl):1

Total Volume(µl):10

We transformed our plasmid into E.coli with a protocol.
We used invitrogen purification kit to get our plasmid. We sent genes to sequencing and sequencing data should be available on their link. Polymerase Chain Reaction of GFP from pKScGFP For 1.0µl of DNA we added, • 1.0µl of forward primer • 1.0µl of reverse primer • 2.5µl of 10X PCR Buffer • 0.5µl of MgSO4 • 0.5µl of dNTP mix • 18.375µl of ddH2O • 0.125µl of Taq polymerase Parameters in thermal cycler were chosen as below: Denaturation is 95oC for 4min Annealing is 30 cycles: • 95oC 1min • 65oC 1min • 72oC 2min Extension 72oC for 10min 4oC hold GFP fusion GFP fusion with nfsI gene on our Biobrick is our plannned future work.