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

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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.

Restriction enzyme assays. Lane1:Ladder. Lane2:Plasmid pS1C3. Lane3:Plasmid harboring promoter J23101 (SpeI/PstI).Lane4:P0451 colony 6 (EcoRI/SpeI) .Lane5: K098991 colony 8 (XbaI/PstI).
  • Ligation mixture 1
Reactive Quantity
DNA inserts (K098991+ P0451) 4μL each
DNA plasmid (pSB1C3) 2μL
Buffer 10X T4 Ligase 2μL
Enzyme T4 Ligase 1μL
HPLC Up to a final volume of 20 μL of the mixture (7μL)


  • Ligation mixture 2
Reactive Quantity
DNA inserts (K098991+ P0451) 5μL each
DNA plasmid (pSB1C3) 3μL
Buffer 10X T4 Ligase 2μL
Enzyme T4 Ligase 1μL
HPLC Up to a final volume of 20 μL of the mixture (4μL)


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
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.