BioFilm Track
- Grew 2 sub-cultres in 10ml of staph + B2 (3ml) for OD curve and staph competent cell. Cell obtained
from glycerol stocks
- Placed in AMBL Rotator (32C, 180rpm)
- Start: 3:30pm (July 05,10)
- End: 9:30am ( July 06, 10)
Misc
- Poured Amp plates (18 plates)
- Placed 2 ON E.coli competent cells in AMBEl rotator (32C, 180rpm)
- Autoclaved 500ml LB Broth media
- Autoclaved 600ml B2 media
dspB Track
Marianne and Vicki
Colony PCR
- Protocol: Used 'colony PCR' in common protocols
- Colony PCR for individual colonies:
- 11T,12T
- 5 colonies from each plate; therefore, 10 PCRs in total
Table 1. Master Mix
REAGENT | 1 RXN VOLUME (uL) | MASTER MIX (uL) |
10x rxn buffer | 2.5 | x13 | 32.5 |
10uM FW primer (G1004) | 2 | x13 | 26 |
10uM RE primer (G1005) | 2 | x13 | 26 |
10mM dNTP | 3 | x13 | 39 |
sdH2O | 15.3 | x13 | 198.9 |
Taq polymerase | 0.2 | x13 | 2.6 |
Total | 25 | | 235 |
Table 2. PCR tubes
11T1 | 11T2 | 11T3 | 11T4 | 11T5 | H2O control (W) |
12T1 | 12T2 | 12T3 | 12T4 | 12T5 |
- For each respective tube, picked colonies numbered 3,5,7,9,11 in order and added to each microcentrifuge tube in ascending order. Ex. Colony #3 from plate 11T into 11T1.
PCR Cycles:
- 94C @ 30 sec
- 56C @ 30 sec
- 68C @ 2 min
- 68C @ 20 min
- Start PCR: 1351
- End PCR: 1603
Gel verification
- Protocol: gel verification protocol in iGEM training manual
Table 3. Gel orientation
layer 1 | 11T1 | 11T2 | 11T3 | 11T4 | 11T5 | 12T1 | 12T2 | 12T3 | 12T4 | 12T5 | 100bp ladder | W | 10T1 | 10T2 | 10T3 | 10T4 | 10T5 | 10T6 | 10T7 | 10T8 |
layer 2 | 10T9 | 10T10 | 1-T11 | 10T12 | 10T13 | 10T14 | 100bp ladder | 10T15 | 10T16 | 10T17 | 10T18 | 10T19 |
- Machine conditions: 110V, 40 minutes, 0.5X TBE buffer
- DNA: 10uL, loading dye: 2uL
Gel results:
Overnight culture
- Protocol: From iGEM common protocols
- Colonies: 8T3, 6T10, 6T11, 6T14
- Cultures (tubes): 8T3, 6T10, 6T11, 6T14, control (LB broth+chlor)
- Put cultures in 37C turntable in AMBL lab @ 1800
Vicki Ma 18:34, 6 July 2010 (EDT)
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