| 1. Plasmid Prep: Plasmid prep from the 2mL cultures made from picked colonies of (a) TetR/p22mnt + RFP + Term, (b) Lac/LAMBDA + GFP 1, (c) Lac/LAMBDA + GFP 2, (d) Tetpro + LAMBDAcI + term.
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| 2. Spectrophotometry: Spec'ed the plasmid prep products to check for concentration of DNA.
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| 3. Digest Rxn: Run a digest reaction on the plasmid prep products. Follow the chart below:
| Parts | 10x Buffer | BSA | H20 | Insert DNA | RE1 | RE2 | Total
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| L/L PCR | 5.0uL | 05uL | 22.5uL | 20.0uL | 1.0uL, EcoRI | 1.0uL, Pst1 | 50.0uL
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| TetR/p22mnt PCR | 5.0uL | 0.5uL | 22.5uL | 20.0uL | 1.0uL, EcoRI | 1.0uL, Pst1 | 50.0uL
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| BV | 5.0uL | 0.5uL | 37.5uL | 5.0uL | 1.0uL, EcoRI | 1.0uL, Pst1 | 50.0uL
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| Lac/LAMBDA + GFP 1 | 5.0uL | 0.5uL | 12.5uL | 30.0uL | 1.0uL, Spe1 | 1.0uL, Pst1 | 50.0uL
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| Term | 5.0uL | 0.5uL | 37.5uL | 5.0uL | 1.0uL, Xba1 | 1.0uL, Pst1 | 50.0uL
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| L/L + GFP 2 | 5.0uL | 0.5uL | 7.5uL | 35.0uL | 1.0uL, Spe1 | 1.0uL, Pst1 | 50.0uL
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| 4. Vector Dephosphorylation of Digest Products: Dephosphorylated the BV, Lac/LAMBDA + GFP 1, and Lac/LAMBDA + GFP 2. Did not dephosphorylate the PCR products or the terminator. Used 1uL of antarctic phosphatase and 5.6uL of antarctic phosphatase buffer. 30 mins @ 37C in an incubator, then 15 minutes of heat inactivation.
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| 5. Run a gel on Digest Terminator Product: Using 1% agarose gel, check for correct amount/type of DNA bands. Only run terminator on gel. Will most likely not visualize band because terminator is approx. 40 bp's long.
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| 6. Gel purification: The terminator band was cut out of the electrophoresis gel and purified out using QIAGEN's QuickSpin gel purification protocol. DNA used in ligation below.
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7. Ligations: Ligated the digested, dephosphorylated, and gel purified products. After following the procedure/table of ligations below, we stored the ligated products @ -20C O/N to then transform tomorrow into competent cells. Folow the ligation table below:
| Parts | 10x Buffer | T4 DNA Ligase | Base Vector | Insert DNA | H20 | Total
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| BV + L/L PCR | 4.0uL | 4.0uL | 1.0uL | 25.0uL | 6.0uL | 40.0uL
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| BV + T/P | 4.0uL | 4.0uL | 1.0uL | 25.0uL | 6.0uL | 40.0uL
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| L/L : GFP 1 + Term | 4.0uL | 4.0uL | 4.0uL | 25.0uL | 3.0uL | 40.0uL
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| L/L : GFP 2 + Term | 4.0uL | 4.0uL | 4.0uL | 25.0uL | 3.0uL | 40.0uL
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| 8. Sequencing Rxns: Send in sequences. Follow the mixture below, which was sent in today:
| Parts | H20 | DNA | primer
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| TetR/p22mnt + RFP + Term | --- | 8.8uL | 3.2uL
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| TetR/p22mnt + RFP + Term | --- | 8.8uL | 3.2uL
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| L/L + GFP 1A | --- | 8.8uL | 3.2uL
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| L/L + GFP 1E | --- | 8.8uL | 3.2uL
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| Tetpro + LAMBDAcI + TermA | --- | 8.8uL | 3.2uL
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| Tetpro + LAMBDAcI + TermB | --- | 8.8uL | 3.2uL
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| L/L + GFP 2A | --- | 8.8uL | 3.2uL
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| L/L + GFP 2B | --- | 8.8uL | 3.2uL
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