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.

Results restriction enzyme assays
Quantity loaded 3μL



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


 * Ligation for plasmid pSB1C3: Restriction enzymes EcoRI and PstI.

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

Procedure

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

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


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