IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/06

{| width="800"
 * style="background-color: #EEE"|[[Image:TeamLogo.jpg|350px]] UNAM-Genomics-Mexico team
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

Working on cI inverter construction and fusion to pSB1C3 backbone. LovTAP repressor activity:reporter system
Once the parts K098991 (cI regulated promoter+RBS+GFP) and P0451(RBS+cI repressor) were correctly digested, I am going to ligate them in plasmid pSB1C3.

Ligation Procedure: P0451 + K098991 to plasmid pSB1C3
1. Prepare the ligation mixture taking into account the quantity of the DNA insertS -K098991 and P0451- and the receiver DNA -plasmid pSB1C3-. This can be check in the following gel. Quantity loaded 3μL.




 * Ligation mixture 1


 * Ligation mixture 2

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 the whole cI inverter, if the ligation was correctly done.

Working on J23101 promoter: Ligations to P0451, LuxY and Lumazine
Once the plasmid harboring the J23101 promoter was correctly digested with the enzymes SpeI and PstI, I have started the dephosphatation reaction in order to prepare it for ligation with P0451, 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.


 * }