IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/27

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Working on LuxYsynthesized from Mr.Gene: Preparation for Ligation

Once the PCR reactions for LuxY 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:

LuxY Restriction enzyme reactions:Preparation for Ligation

  • Ligation for promoters (J23101 and J23102): Restriction enzymes XbaI and PstI.
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

Results restriction enzyme assays

Quantity loaded 3μL

Restriction enzyme assays. Lane1:Ladder. Lane6: LuxY PCR amplified product from Mr.Gene (EcoRI/PstI). Lane7:LuxY PCR amplified product from Mr.Gene(XbaI/PstI). The other lanes are samples from other experiments

The sizes of the digested products are around the expected lenght for each part.


  • Ligation for plasmid pSB1C3: Restriction enzymes EcoRI and PstI.
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:Desactivate the reaction at 80°C during 20 min.

Getting more sample:Working on J23102 promoter: Ligations to P0451, LuxY and Lumazine

Once the plasmid harboring the J23102 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.