| 1. Pick Colonies from BV:Lac/LAMBDA (dual promoter and base vector): Picked colonies and placed BV:dualpromoters in 2mL LB cultures to incubate @ 37C for 8-12 hours. (Also make one 'control' w/ 2mL LB and pipet tip but no colony on it).
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| 2. Plasmid Prep (1) BV:Tet/p22:RFP and (2) BV:Tetpromoter:LAMBDAcIgene: Using QIAmini prep guidelines - prepped 5 samples of BV:TetR/p22:RFP and 5 samples of BV:TetRpromoter:LAMBDAcIgene. Steps: Pour LB culture into tube. Spin tube and remove supernatant. Resuspend pelleted bacterial cells in 250uL Buffer P1 Resuspension buffer. Add 250uL Buffer P2 Lysis buffer and invert 4-6 times. Add 350uL of Buffer N3 Neutralization buffer and invert 4-6 times. Centrifuge for 10 minutes @ 13,000 rpm's. Put supernatants in to spin column, centrifuge for 30-60 seconds (spin column binds to DNA and everything else flows through). Discard flow through. Add 500.0uL of Buffer PB binding buffer, centrifuge for 30-60 seconds, discard flow through. Add 750.0uL Buffer PE wash buffer, centrifuge for 30-60 seconds, discard supernatants and centrifuge for an additional 30-60 seconds. Place spin column in microcentrifuge tube, and elute DNA by adding 50uL Buffer EB elution buffer (10mM Tris-Cl, pH 8.5)let stand for 5 minutes and centrifuge.
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| NOTE: Last step of Plasmid Prep was performed differently to obtain a higher concentration of DNA. After elution buffer fell through and flow through had DNA in it so repoured that into spin column a second time to spin again for another 60 seconds to extract more DNA.
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| 3. Spectrophotometry: Spec the 10 plasmid prep samples to check for concentration of DNA.
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| 4. Double Digest: Digest Plasmid Prep products. After following procedure below
| Parts | 10x Buffer | BSA | H20 | DNA | RE 1 | RE 2 | Total
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| BV + T/P + RFP (1) | 5.0uL | 0.5uL | 2.5uL | 40.0uL | 1.0uL, Spe1 | 1.0uL, Pst1 | 50.0uL
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| BV + T/P + RFP (2) | 5.0uL | 0.5uL | 2.5uL | 40.0uL | 1.0uL, Spe1 | 1.0uL, Pst1 | 50.0uL
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| BV + Tetpro. + LAMBDAcI (1) | 5.0uL | 0.5uL | 2.5uL | 40.0uL | 1.0uL, Spe1 | 1.0uL, Pst1 | 50.0uL
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| BV + Tetpro. + LAMBDAcI (2) | 5.0uL | 0.5uL | 2.5uL | 40.0uL | 1.0uL, Spe1 | 1.0uL, Pst1 | 50.0uL
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| Terminator | 5.0uL | 0.5uL | 40.5uL | 2.0uL | 1.0uL, Xba1 | 1.0uL, Pst1 | 50.0uL
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| 5. Vector Dephosphorylate: Vector Dephosphorylate the base vector by adding 1uL Antarctic phosphatase and 5uL of Antarctic Phosphatase buffer.
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| 5. Run a gel: Run gel with (1) BV:Tet/p22:RFP and (2) BV:TetRpromoter:LAMBDAcI. Run for 60 minutes @ 80 volts. Results: NO DNA bands found.
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| 6. Plasmid Prep the Lambda/LacI:BV cultures: Follow same steps as step 2.
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