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 + promoters: Ligation to backbones: Restriction enzymes EcoRI and PstI.

Plasmids used:

LovTAP + J23117 isolated form colony 6.

LovTAP + J23105 isolated form colony 5.

LovTAP + J23114 isolated form colony 4.

LovTAP + J23102 isolated form colony 5.

Reactive Quantity
Plasmid DNA 15μL
Buffer 2 4μL
BSA 1μL
Enzymes 2μL for each one.
HPLC Up to a final volume of 40 μL of the mixture

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.

LovTAP + promoters: Restriction enzyme assay with EcoRI and PstI. Lane1:Ladder.Lane4: Plasmid LovTAP+J23102 colony 5.Lane5:Plasmid LovTAP+J23105 colony 5.Lane6: Plasmid LovTAP+J23114 colony 4.Lane7:Plasmid LovTAP+J23117 colony 6.Lane8:LovTAP Mr.Gene PCR amplified product and digested. The other lanes are samples from other experiments.

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).

Dephosphatation mixture

Reactive Quantity
DNA 15μL
Buffer 10X 3μL
Dephosphatase Enzyme 1.5μL
HPLC Up to a final volume of 30 μL of the mixture (10.5μL)


Procedure

1.Incubate the samples at 37°C during 20 min.

2.Incubate the samples at 65°C during 10 min.