IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/26

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Working on LovTAP fused to promoters, BBa_K098991,LuxY and Lumazine:Ligations to backbones pSB3K3 ans pSB1C3
Once the plasmids pSB3K3 and pSB1C3 were correctly digested with the enzymes EcoRI and PstI, I have started the dephosphatation reaction in order to prepare them for ligation with promoters-LovTAP,BBa_K098991(cI regulated promoter+RBS+GFP), LuxY and Lumazine constructions.


 * Dephosphatation mixture

Procedure

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

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

Repeated experiment:Working on LovTAP fused to promoters, Lumazine and K098991:Ligations to backbone pSB3K3 and pSB1C3
Due to the first attempt to LovTAP fused to promoters, Lumazine and K098991 to backbones pSB1C3 and pSB3K3 failed, I am going to repeat the experiment.

Ligation Procedure:LovTAP fused to promoters, Lumazine and K098991 with backbones pSB1C3 and pSB3K3
1. Prepare the ligation mixture taking into account the quantity of the DNA insert -LovTAP fused to promoters, Lumazine and K098991- and the receiver DNA - Backbones pSB3K3 and pSB1C3 -. This can be check in the following gels.






 * Ligation mixture using plasmid pSB3K3


 * Ligation mixture using plasmid pSB1C3

2. Incubate the sample at 16°C overnight.

3. Transform the cells. Click here for the protocol.

4. Culture the cells in the proper selective medium, in our case LB + Kanamycin.

5. Incubate the petri dishes at 37°C overnight.

6. Re-culture the resultant colonies in the proper selective medium; incubate them at 37°C overnight.

7. Analyze the colonies with Colony PCR  to confirm that they contain the correct ligation

The primers that I am using are:

Forward (5'->3'): Preffix primer.

Reverse:(5'->3'): Suffix primer.

These primers would amplify LovTAP ligated to each promoter and BBa_K098991, if the ligation was correctly done.

Getting more sample: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  again, 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.

The reactions were incubated at 37°C overnight.

Note: Inactivate the enzymes at 80°C during 20 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.


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