IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/01
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Working on LovTAP fused to promoters: Ligation to backbone pSB3K3 for characterization and pSB1C3 for DNA submission
Once the ligations of LovTAP with promoters were confimed, I am going to digest them with EcoRI and PstI in order to fuse them to backbones pSB3K3 and pSB1C3.
LovTAP + J23117 isolated form colony 6.
LovTAP + J23105 isolated form colony 5.
LovTAP + J23114 isolated form colony 4.
LovTAP + J23102 isolated form colony 5.
The reactions were incubated at 37°C overnight.
Note: Inactivate the enzymes at 80°C during 10 min.
Results: Restriction enzyme assay Preparing LovTAP and promoters for plasmids backbone ligations
According to the next image, it seems that all the LovTAP ligations with promoters, were correctly digested because the bands obtained are around the expected size of the LovTAP gene (889nt) plus the promoters lenght.
These digestions will be fused to plasmids pSB3K3 and pSB1C3.
Working on cI inverter promoter: Dephosphatation of the strong promoter J23102 for ligation
Once the plasmid harboring the promoter J23102 were correctly digested with the enzymes SpeI and PstI, I have started the dephosphatation reaction in order to prepare them for ligation with the part BBa_P0451 (RBS+cI repressor).
1.Incubate the samples at 37°C during 20 min.
2.Incubate the samples at 65°C during 10 min.