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From 2013.igem.org

Contents 1 Digesting pSB1C3 backbone 2 Digestion of the TOD genes PCR products 3 Running the backbone and RFP on gel 4 Digestion of the pGEM plasmid extracted on the 13/09/13

Digesting pSB1C3 backbone

• Digesting for ligation with the already digested Tod operon PCR products
• Following the iGEM official protocol
• Using EcoRI-HF and PstI-HF
• Exluding the DpnI from the master mix and adjusting the volume of water accordingly
• Protocol can be found at: [http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones]

Digestion of the TOD genes PCR products

The PCR products of TODX, TODF and TOBG were digested with the enzymes EcoRI-HF and PstI-HF.

Protocol

Sample	DNA volume (ul)	cutsmart buffer (ul)	EcoRI (ul)	PstI (ul)	dH2O (ul)
TODX	8.7	2	0.5	0.5	8.3
TODF	8.3	2	0.5	0.5	8.7
TOBG	8	2	0.5	0.5	9

• The samples were incubated for 1 hour at 37C.
• Heat kill the enzymes at 80C for 20 minutes.

Running the backbone and RFP on gel

• on 1% gel to check if the gel extraction worked.

Digestion of the pGEM plasmid extracted on the 13/09/13