dspB Track
Restriction Digest Chlor backbone psb1C3
- Protocol: biobrick (digesting)
Restriction Digest Supermix
REAGENTS | 1 RXN VOLUME (uL |
Buffer 2 | 5 |
BSA | 0.5 |
ddH2O | 37 |
Total | 42.5 |
- Add: 5uL of backbone
- Enzymes: EcoRI and SpeI (1uL each to each tube)
Transformation
- Transform ligation mixes from Aug 13 2010
- Tubes (ligation mixes): 1,2,3,4,5,6,7,8,11,12,13,14,15,AA,AB,TA,TB,CA
- Protocol: Common protocols - SOP
- Changes: Add 10uL ligation mixes instead of 1uL
Note: 14,15, AA,AB,TA,TB,CA were transformed with 2uL of ligation mix
- Alina ligated His (7) and No-his (23). Will also transform: also add 2uL of his + no-his ligation mixes
- Transformants put in 37C incubator at 1450, taken out at 1617
Control Experiment
- RD Chlor (psb1C3) and Amp (psb1A3) @ EcoRI, XbaI, PstI cut sites
- Protocol: RD Protocol (biobrick)
Restriction Digest Supermix
REAGENTS | 1 RXN VOLUME (uL |
Buffer 2 | 5 |
BSA | 0.5 |
ddH2O | 37 |
Total | 42.5 |
- Add DNA: 5uL of backbone
- Enzymes: EcoRI, XbaI, PstI (1 uL each to each tube)
QS Track
- Like the dspB track, a bacterial mat grew when transformants were spread on chloramphenicol plates.
- A bacterial mat also grew when transformants were spread on LB + 20 μL chloramphenicol (1000X).
- This suggests a possible problem with the chloramphenicol aliquots (1000X) used.
- Marianne remade the chloramphenicol aliquots using 3 different sources of chloramphenicol.
- Phillip made chloramphenicol plates (20) using Marianne's C3 aliquots (chlor source: last year's parafilm sealed bottle)
- From yesterday's leftover ligation mixtures, 5 μL was transformed again by Vicki (following UBC iGEM procedures).
- Also started an overnight culture of pcN33. Host is Staph aureus RN4220. Used LB broth (no erm in lab).
- Made an antibiotic test of possible problematic chloramphenicol aliquots mentioned earlier. 10 μL of chloramphenicol aliquot was added to 10 mL LB. A stab of stock DH5(alpha)was inoculated in this medium. Expect no growth.
Biofilm Track
Melody
Biofilm Growth Protocol Day 1 (100823M+E)
- Performed Day 1 of the Biolfilm Protocol
- O/N cultures of Control, RN4220 and 8325-4 in 5mL of TSB
- Into incubator @ 1425
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