IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/07
UNAM-Genomics-Mexico team | Main project page Previous entry Next entry | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Working on J23101 promoter: Ligation to P0451Once the plasmid harboring the J23101 promoter was correctly dephosphatated, I have started the ligation procedure with P0451 (RBS+cI repressor). Ligation Procedure:Promoter J23101 with P0451 (RBS+cI repressor)1. Prepare the ligation mixture taking into account the quantity of the DNA insert -P0451 (RBS+cI repressor)- and the receiver DNA -plasmid harboring the promoter J23101-. This can be check in the following gel.
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 P0451:RBS+cI repressor plus promoter J23101, if the ligation was correctly done. Working on Lumazine synthesized from Mr.Gene: Preparation for LigationOnce the PCR reactions for Lumazine gene were successfully done, I purified both PCR mixures using the High Pure PCR Product Purification kit from Roche. The purified product will be digested for the following ligations. Lumazine Restriction enzyme reactions:Preparation for Ligation
The reactions were incubated at 37°C overnight. Note:Desactivate the reaction at 80°C during 10 min. Ligation Procedure: Lumazine/LuxY with plasmid pSB1C3 and promoters J23101/J231021. Prepare the ligation mixture taking into account the quantity of the DNA inserts -Lumazine/LuxY - and the receiver DNA -plasmid pSB1C3 and plasmids haboring the promoters J23101/J23102-. This can be check in the following gel. Quantity loaded 3μL.
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.
|