Lethbridge/Notebook
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===July 25=== | ===July 25=== | ||
GFP repressor (TetR) and LacI inhibited promoter transformed successfully | GFP repressor (TetR) and LacI inhibited promoter transformed successfully | ||
| - | RFP with promoter, RBS, and terminators transformed but does not emit RF | + | RFP with promoter, RBS, and terminators transformed but does not emit RF under UV light and plasmid too small, will have to build from five subparts. |
| - | + | ||
GFP, LacI, and four subparts of RFP left to go | GFP, LacI, and four subparts of RFP left to go | ||
Revision as of 17:40, 25 August 2007
Contents |
July 07 2007
Attempted Transformation of 4 Biobricks.
1. BBa_J5526 - Plate 3 well 6F - RFP 2. BBa_I13522 - Plate 2 well 15H - GFP 3. BBa_R0011 - Plate 1 well 7M - LacI inhibited promoter 4. BBa_PO440 - Plate 1 well 21K - GFP repressor
Protocol
1. Add 15uL deionized H2O to well 2. Add 1 uL plasmid to 25uL DH5alpha 3. Incubate on ice for 30min 4. Heat shock without shaking for 20sec 5. Place on ice for 2min 6. Add 0.5mL LB, incubate for 1hr 7. Plate 100uL on amp+ LB plates, incubate overnight at 37 C
July 14 2007
RFP and GFP repressor plates had colonies (RFP had only 1 colony). GFP and promoter did not.
July 16 2007
Repeated transformation of GFP, RFP, and promoter, with addition of a cell concentraion step and more cells/plasmid. (Revisions to protocol in bold.)
Protocol
1. Add 35uL deionized H2O to well 2. Add 5uL plasmid to 50uL DH5alpha 3. Incubate on ice for 30min 4. Heat shock without shaking for 20sec 5. Place on ice for 2min 6. Add 0.5mL LB, incubate for 1hr 7. Spin down cells gently, remove 350uL supernatant and resuspend (gently!) pellet 8. Plate 100uL on amp+ LB plates, incubate overnight at 37 C
Picked colonies from RFP and GFP repressor and inoculated amp+ LB broth
July 17 2007
GFP and promoter had colonies. RFP did not. Performed plasmid prep on RFP and GFP repressor cultures according to QIAprep Miniprep Handbook 2nd Ed. Nov '05 rip out bench protocol for microcentrifuge.
July 18 2007
Ran extracted plasmids on a gel
RFP appears to be too small, and cells do not show red flourescence under UV light. Since part appears not to be working, will build RFP part from 5 subparts.
July 23 2007
Attempted transformation of 6 biobricks
1. BBa_I13522 - plate 2 well 15H - GFP - had to redo GFP because accidentally transformed plate 1 well 15H the first time 2. BBa_C0012 - plate 1 well 5A - Lac I 3. BBa_B0034 - plate 1 well 3O - RBS 4. BBa_E1010 - plate 2 well 15M - RFP 5. BBa_B0010 - plate 2 well 3P - T1 6. BBa_B0012 - plate 1 well 1C - T2
Protocol
1. Add 15uL deionized H2O to well 2. Add 1 uL plasmid to 25uL DH5alpha 3. Incubate on ice for 30min 4. Heat shock without shaking for 20sec 5. Place on ice for 2min 6. Add 0.5mL LB, incubate for 1hr 7. Spin 13000 rpm for 1min, remove 400uL supernatant and resuspend 7. Plate 100uL on amp+ LB plates, incubate overnight at 37 C
July 24
Note book created (All previous entries are transcribed from paper lab notebook)
All transformations worked except LacI. Picked two colonies from each and cultured in 5mL amp+ LB, incubated overnight at 37 C.
July 25
GFP repressor (TetR) and LacI inhibited promoter transformed successfully RFP with promoter, RBS, and terminators transformed but does not emit RF under UV light and plasmid too small, will have to build from five subparts. GFP, LacI, and four subparts of RFP left to go
July 27
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