| 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.
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| 2. Spectrophotometry: Excellent results from spec'ing new base vector plasmid. Very high concentration of DNA, which was 430 ng/uL.
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| 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
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| BV | 5.0uL | 0.5uL | 30.8uL | 11.7uL | EcoRI, 1.0uL | Pst1, 1.0uL | 100.62 ng/uL
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| TetR Pro. 1 | 5.0uL | 0.5uL | 35.5uL | 7.0uL | EcoRI, 1.0uL | Pst1, 1.0uL | 23.8 ng/uL
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| Tet R Pro. 2 | 5.0uL | 0.5uL | 35.5uL | 7.0uL | EcoRI, 1.0uL | Pst1, 1.0uL | 23.8 ng/uL
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| LacI Pro. 1 | 5.0uL | 0.5uL | 41.5uL | 1.0uL | EcoRI, 1.0uL | Pst1, 1.0uL | 168.0 ng/uL
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| LacI Pro. 2 | 5.0uL | 0.5uL | 41.5uL | 1.0uL | EcoRI, 1.0uL | Pst1, 1.0uL | 168.0 ng/uL
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| Lac/Lam 1 | 5.0uL | 0.5uL | 28.5uL | 14.0uL | EcoRI, 1.0uL | Pst1, 1.0uL | 9.8 ng/uL
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| Tet/p22 1 | 5.0uL | 0.5uL | 34.0uL | 8.5uL | EcoRI, 1.0uL | Pst1, 1.0uL | 20.4 ng/uL
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| Tet/p22 2 | 5.0uL | 0.5uL | 34.0uL | 8.5uL | EcoRI, 1.0uL | Pst1, 1.0uL | 20.4 ng/uL
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| 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
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| BV | 1.0uL | 5.1uL
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| 5. Gel electrophoresis: After heat inactivating the enzyme @ 65C for 15 minutes, we ran a gel with the BV and double digest proucts.
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| 6. Gel Purified: Cut out bands from gel and purified those bands (which contain the DNA wanted).
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| 7. Spetrophotometry: Spec the purified gel DNA to measure concentration of DNA in eight parts.
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