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From 2013.igem.org

Contents 1 Digesting pSB1C3 backbone 2 Ligation of digested pSB1C3 backbone to Tod F, X and Tob G 3 Transformation of TOD genes ligations done on the 02/09/13 using the pGEM-T Vector System I-A3600 (changes were made to the protocol) 4 Digestion of the TOD genes PCR products 5 Running the backbone and RFP on gel 6 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]

Ligation of digested pSB1C3 backbone to Tod F, X and Tob G

• Using Promega protocol to which changes were made

Reagents	Tod F	Tod X	Tob G	Positive control	Background control
2x Rapid Ligation buffer	5ul	5ul	5ul	5ul	5ul
pSB1C3 digest (50ng)	2ul	2ul	2ul	2ul	2ul
PCR product	3.3ul	3ul	3.8ul	-	-
Control insert DNA	-	-	-	2ul	-
T4 DNA ligase	1ul	1ul	1ul	1ul	1ul
Deionized water for final volume of 10ul	-	-	-	-	1ul

Transformation of TOD genes ligations done on the 02/09/13 using the pGEM-T Vector System I-A3600 (changes were made to the protocol)

• Centrifuge the ligations reactions briefly.
• Add 5ul of each ligation reaction to a sterile 1.5ml
• Thaw the competent cells on ice. Mix the cells by gently flicking the tube.
• Carefully transfer 100ul of the competent cells to the ligation reaction tubes.
• Incubate the tubes on ice for 20 minutes
• Heat-shock the cells for 45-50 seconds in a water bath at exactly 42C degrees. Do not Shake. Immediately return the tubes to ice for 2 minutes.
• Add 950ul of room temperature SOC medium to the ligation reaction transformations. incubate for 1.5 hours at 37C with shaking.

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