| 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. Run a gel: Using 1% agarose gel, check for correct amount/type of DNA bands.
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| 5. 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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| 6. 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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