| 1. Plasmid prep dual promoters: (1) Tet & P22mnt, (2) LacI & LAMBDAcI.
|
| 2. Model: Figure out why have such a low GFP output when run model.
|
| 3. Sequence L5, L6-L9
|
| 4. Re-transform RFP, TetR promoter, terminator, and MCherry because no cell growth on plates. Place in 2mL LB cultures, allow growth in incubator @37C for 2 hours. Plate samples on Ampicillin resistant plates. Allow growth O/N.
|
| 5. Discuss problems with sequences
|
| 6. Double digest of all ligations: from previous days. Do a double digest to cut the components linearly, which will then be able to run through a gel to check if correct DNA
is present. Follow the table below:
| Parts | 10x Buffer | BSA | H20 | DNA | RE 1 | RE 2
|
| L2a, LAMBDAcI:Term | 5.0uL | 0.5uL | 32.5uL | 10.0uL | EcoRI | Xba1
|
| L2b, LAMBDAcI:Term | 5.0uL | 0.5uL | 32.5uL | 10.0uL | EcoRI | Xba1
|
| L3i, Pro:LAMBDAcI | 5.0uL | 0.5uL | 32.5uL | 10.0uL | Pst1 | Spe1
|
| L3j, Pro:LAMBDAcI | 5.0uL | 0.5uL | 32.5uL | 10.0uL | Pst1 | Spe1
|
| L4b, LAMBDAcI:Term | 5.0uL | 0.5uL | 40.5uL | 2.0uL | EcoRI | Xba1
|
| L4c, LAMBDAcI:Term | 5.0uL | 0.5uL | 39.5uL | 3.0uL | EcoRI | Xba1
|
| L4d, LAMBDAcI:Term | 5.0uL | 0.5uL | 39.5uL | 3.0uL | EcoRI | Xba1
|
| L4e, LAMBDAcI:Term | 5.0uL | 0.5uL | 39.5uL | 3.0uL | EcoRI | Xba1
|
| L4g, LAMBDAcI:Term | 5.0uL | 0.5uL | 39.5uL | 3.0uL | EcoRI | Xba1
|
| L4h, LAMBDAcI:Term | 5.0uL | 0.5uL | 39.5uL | 3.0uL | EcoRI | Xba1
|
| L4i, LAMBDAcI:Term | 5.0uL | 0.5uL | 37.5uL | 5.0uL | EcoRI | Xba1
|
| L4j, LAMBDAcI:Term | 5.0uL | 0.5uL | 32.5uL | 10.0uL | EcoRI | Xba1
|
| L5c, GFP:Term | 5.0uL | 0.5uL | 40.5uL | 2.0uL | EcoRI | Xba1
|
| L6a, Pro:LAMBDAcI:Term | 5.0uL | 0.5uL | 39.5uL | 3.0uL | EcoRI | Xba1
|
| L6b, Pro:LAMBDAcI:Term | 5.0uL | 0.5uL | 41.5uL | 1.0uL | EcoRI | Xba1
|
| L6e, Pro:LAMBDAcI:Term | 5.0uL | 0.5uL | 39.5uL | 3.0uL | EcoRI | Xba1
|
| L6d, Pro:LAMBDAcI:Term | 5.0uL | 0.5uL | 39.5uL | 3.0uL | EcoRI | Xba1
|
| L7a, Pro:LAMBDAcI:Term | 5.0uL | 0.5uL | 38.5uL | 4.0uL | EcoRI | Xba1
|
| L7b, Pro:LAMBDAcI:Term | 5.0uL | 0.5uL | 39.5uL | 3.0uL | EcoRI | Xba1
|
| L7d, Pro:LAMBDAcI:Term | 5.0uL | 0.5uL | 37.5uL | 5.0uL | EcoRI | Xba1
|
| L8a, Pro:LAMBDAcI:Term | 5.0uL | 0.5uL | 39.5uL | 3.0uL | EcoRI | Xba1
|
| L9a, Pro:LAMBDAcI:Term | 5.0uL | 0.5uL | 41.5uL | 1.0uL | EcoRI | Xba1
|
| L9c, Pro:LAMBDAcI:Term | 5.0uL | 0.5uL | 40.5uL | 2.0uL | EcoRI | Xba1
|
| L9d, Pro:LAMBDAcI:Term | 5.0uL | 0.5uL | 41.5uL | 1.0uL | EcoRI | Xba1
|
|
| 7. Gel Electrophoresis: Performed on double digest reaction from above. Refer to picture below for results.
|
|
|
| Left to Right: Ladder, --, L9E, L9C, L9A, L8A, L7D, L7B, L7A, L6E, L6D, L6B, L6A, L5C, --, Ladder, L4j, L4i, L4h, L4g, L4d, L4e, L4c, L4b, L3j, L3i, L2b, L2a, --, Ladder
|
"
