| 1. Analyze gel results from 07-17-2008: Send in select samples from the gel for sequencing. Send in reaction mixture containing template DNA and primers that will bind to it, and thus allow to sequence. Sequencing L3i, L3j, L4b, L4i, L5c, L6a, L6b, L6d, L6e, L7a, L7b, L7d, and L8a.
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| 2. Problem: No growth on plates with MCherry, RFP, Terminator, and TetR promoter.
Solution: Re-transform (again) paper DNA with MCherry, RFP, Term., and TetR.
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| 3. Double Restriction Digest: Double digest dually repressed promoters; (1) Lac/LAMBDAcI, (2) TetR/p22mnt, along with reporter genes; GFP and YFP. Incubate @ 37C for 2-20 hrs. Heat inactivate for 15 mins @ 65C. Follow the table below:
| Parts | 10x Buffer | BSA | H20 | DNA | RE 1 | RE 2
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| (A) Lac/LAMBDAcI Promoters | 5.0uL | 0.5uL | 13.5uL | 29.0uL | 1.0uL, EcoRI | 1.0uL, Spe1
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| (B) Lac/LAMBDAcI Promoters | 5.0uL | 0.5uL | 9.5uL | 33.0uL | 1.0uL, EcoRI | 1.0uL, Spe1
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| (A) TetR/p22mnt Promoters | 5.0uL | 0.5uL | 34.1uL | 8.4uL | 1.0uL, EcoRI | 1.0uL, Spe1
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| (B) TetR/p22mnt Promoters | 5.0uL | 0.5uL | 34.1uL | 8.4uL | 1.0uL, EcoRI | 1.0uL, Spe1
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| GFP:Term | 5.0uL | 0.5uL | 20.5uL | 22.0uL | 1.0uL, EcoRI | 1.0uL, Xba1
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| RFP will not be used because it has not been properly transformed yet. Will result in:
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| Lac/LAMBDAcI:GFP:Terminator
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| Tet/p22mnt:YFP:Terminator
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| 4. Ligate Digested products: Ligated -
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| a. Lac/LAMBDAcI promoter + GFP:Term + BaseVector
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| b. TetR/p22mnt promoter + YFP + BaseVector
| Parts | 10x Buffer | H20 | Prom | Reporter | T4 Ligase
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| (a) Lac/LAMBDAcI : GFP : Term | 3.0uL | 6.0uL | 15.0uL | 5.0uL | 1.0uL
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| (b) Lac/LAMBDAcI : GFP : Term | 3.0uL | 6.0uL | 15.0uL | 5.0uL | 1.0uL
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| TetR/p22mnt : YFP | 3.0uL | 6.0uL | 15.0uL | 5.0uL | 1.0uL
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| 5. Work on model: Model allows us to run potential reactions computationally. This will give us an idea of whether or not there will be errors in the rxns and what outputs will result.
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| 6. Sequencing Rxns: Ran the parts through a gel on 07-17-08, we selected the lanes with the best outcomes. Send into Biomedical Genomics Center to be sequenced -
| Part | Primer | DNA | H20 | KJL
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| (a) L3i | 3.2uL, LAMBDAcI F | 5.0uL | 3.8uL | 142
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| (b) L3i | 3.2uL, BaseVec. F | 5.0uL | 3.8uL | 143
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| (a) L3j | 3.2uL, LAMBDAcI F | 5.0uL | 3.8uL | 144
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| (b) L3j | 3.2uL, BaseVec. F | 5.0uL | 3.8uL | 145
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| (a) L4b | 3.2uL, LAMBDAcI F | 2.4uL | 4.6 uL | 146
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| (b) L4b | 3.2uL, BaseVec. F | 2.4uL | 4.6uL | 147
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| (a) L4i | 3.2uL, LAMBDAcI F | 6.0uL | 2.8uL | 148
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| (b) L4i | 3.2uL, BaseVec. F | 6.0uL | 2.8uL | 149
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| (a) L5c | 3.2uL, LAMBDAcI F | 3.5uL | 5.3uL | 150
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| (b) L5c | 3.2uL, BaseVec. F | 3.5uL | 5.3uL | 151
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| (a) L6a | 3.2uL, LAMBDAcI F | 5.0uL | 3.8uL | 152
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| (b) L6a | 3.2uL, BaseVec. F | 5.0uL | 3.8uL | 153
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| (a) L6b | 3.2uL, LAMBDAcI F | 2.2uL | 6.6uL | 154
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| (b) L6b | 3.2uL, BaseVec. F | 2.2uL | 6.6uL | 155
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| (a) L6d | 3.2uL, LAMBDAcI F | 5.0uL | 3.8uL | 156
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| (b) L6d | 3.2uL, BaseVec. F | 5.0uL | 3.8uL | 157
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| (a) L6e | 3.2uL, LAMBDAcI F | 5.0uL | 3.8uL | 158
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| (b) L6e | 3.2uL, BaseVec. F | 5.0uL | 3.8uL | 159
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| (a) L7a | 3.2uL, LAMBDAcI F | 6.0uL | 2.8uL | 160
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| (b) L7a | 3.2uL, BaseVec. F | 6.0uL | 2.8uL | 161
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| (a) L7b | 3.2uL, LAMBDAcI F | 5.0uL | 3.8uL | 162
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| (b) L7b | 3.2uL, BaseVec. F | 5.0uL | 3.8uL | 163
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| (a) L7d | 3.2uL, LAMBDAcI | 5.0uL | 3.8uL | 164
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| (b) L7d | 3.2uL, BaseVec. F | 5.0uL | 3.8uL | 165
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| (a) L8a | 3.2uL, LAMBDAcI | 5.0uL | 3.8uL | 166
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| (b) L8a | 3.2uL, BaseVec. F | 5.0uL | 3.8uL | 167
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