Minnesota/17 July 2008

From 2008.igem.org

Latest revision as of 20:55, 5 August 2008

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.

Gel from 07-17-2008

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

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