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From 2008.igem.org

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1. Base Vector Plasmid Maxi Prep Base Vector: Grew another culture of different base vector colonies, so now need to plasmid prep the base vector so the DNA/plasmid will be extracted from the rest of the cell.

2. Spectrophotometry: Excellent results from spec'ing new base vector plasmid. Very high concentration of DNA, which was 430 ng/uL.

3. Base Vector & Promoter Double Digest: 50uL Reaction Mixture. Digest incubation for 2 hours @ 37C. Heat inactivate enzyme for 15 minutes @ 65C in a water bath. Follow the table below for pre-incubation steps: Parts 10x Buffer BSA H20 DNA from parts RE 1 RE 2 [DNA] ng/uL BV 5.0uL 0.5uL 30.8uL 11.7uL EcoRI, 1.0uL Pst1, 1.0uL 100.62 ng/uL TetR Pro. 1 5.0uL 0.5uL 35.5uL 7.0uL EcoRI, 1.0uL Pst1, 1.0uL 23.8 ng/uL Tet R Pro. 2 5.0uL 0.5uL 35.5uL 7.0uL EcoRI, 1.0uL Pst1, 1.0uL 23.8 ng/uL LacI Pro. 1 5.0uL 0.5uL 41.5uL 1.0uL EcoRI, 1.0uL Pst1, 1.0uL 168.0 ng/uL LacI Pro. 2 5.0uL 0.5uL 41.5uL 1.0uL EcoRI, 1.0uL Pst1, 1.0uL 168.0 ng/uL Lac/Lam 1 5.0uL 0.5uL 28.5uL 14.0uL EcoRI, 1.0uL Pst1, 1.0uL 9.8 ng/uL Tet/p22 1 5.0uL 0.5uL 34.0uL 8.5uL EcoRI, 1.0uL Pst1, 1.0uL 20.4 ng/uL Tet/p22 2 5.0uL 0.5uL 34.0uL 8.5uL EcoRI, 1.0uL Pst1, 1.0uL 20.4 ng/uL

4. Vector Dephosphorylation: After the digested products' enzyme has been heat inactivated, we dephosphorylated the base vector so the cut ends wouldn't religate. Follow the table below: Parts Antarctic Phosphatase Antarctic Phosphatase Buffer BV 1.0uL 5.1uL

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