| 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.
|
| 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.
|
3. Double 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.
| Parts | 10x Buffer | BSA | H20 | DNA | RE 1 | RE 2
|
| (A) Lac/LAMBDAcI Promoters | 5.0uL | 0.5uL | 13.5uL | 29.0uL | 1.0uL, EcoRI | 1.0uL, Spe1
|
| (B) Lac/LAMBDAcI Promoters | 5.0uL | 0.5uL | 9.5uL | 33.0uL | 1.0uL, EcoRI | 1.0uL, Spe1
|
| (A) TetR/p22mnt Promoters | 5.0uL | 0.5uL | 34.1uL | 8.4uL | 1.0uL, EcoRI | 1.0uL, Spe1
|
| (B) TetR/p22mnt Promoters | 5.0uL | 0.5uL | 34.1uL | 8.4uL | 1.0uL, EcoRI | 1.0uL, Spe1
|
| GFP:Term | 5.0uL | 0.5uL | 20.5uL | 22.0uL | 1.0uL, EcoRI | 1.0uL, Xba1
|
|
RFP will not be used because it has not been properly transformed yet. Will result in:
| Lac/LAMBDAcI:GFP:Terminator
|
| Tet/p22mnt:YFP:Terminator
|
| 5. Ligate Digested products:
|
| 4. 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.
|
"
