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Team:Evry/Notebook/CellCharacterization
From 2014.igem.org
Notebook - Cell Characterization
Preparation of antibiotic stocks
| Antibiotic | Stock concentration | Protocol |
| Kanamycin | 25 mg/mL | Weight 0.56g of Kanamycin powder, solubilization into 20mL of miliQ water. Vortex 5min and filtration with a 200nm filter. Stock : -20°C (aliquots of 1mL) |
| Streptomycin | 100 mg/mL | Weight 1g of Streptomycin powder, solubilization into 20mL of miliQ water. Vortex 5min and filtration with a 200nm filter. Stock : -20°C (aliquots of 1mL) |
| Amplicilin | 100 mg/mL | Weight 1g of Amplicilin powder, solubilization into 20mL of miliQ water. Vortex 5min and filtration with a 200nm filter. Stock : -20°C (aliquots of 1mL) |
| Tetracyclin | 15 mg/mL | Weight 0.6g of Tetracyclin powder, solubilization into 20mL of ethanol 50%. Vortex 5min and filtration with a 200nm filter. Stock : -20°C and hiden from light(aliquots of 1mL) |
| Chloramphenicol | 34 mg/mL | Weight 0.34g of Chloramphenicol powder, solubilization into 10mL of ethanol 100%. Vortex 5min and filtration with a 200nm filter. Stock : -20°C and hiden from light(aliquots of 1mL) |
Tests of antibiotics' stocks - Results
Survivability tests - Results
| Plates | LB Agar only | LB+Cam | LB+Kan | LB+Amp | LB+Erm | LB+Tet |
| E.coli | X | 0 | X | 0 | X | 0 |
There was a problem with the stock of Kanamycine so we made a new one at 25mg/mL.
For the erythrompycine, we learn that E. Coli is not really sensitive to this antibiotic and that it's better to use it with a dilution at 1:100.
Survivability tests - Results
| liquid cultures/Plates | MB | M9 |
| MB | ++++ | ++ |
| M9 | ++ | + |
Any liquid culture can be plated and will grow up. Of course, it works better on MB medium, wich is a rich medium than on M9.
Aug 17
Tests of antibiotics' stocks
Six plates of LB agar were made.
Five of them contained one of those antibiotics in the dilution 1:1000:
(The last one was the control of the growth of our bacteria without antibiotics) We sowed 10µL of a liquid LB culture of Bl21, and we let them incubate overnight at 37°C.
Survivability tests
Two plates of MB 1X and M9 1X were made and divised in two parts. Each plate was sowed with 5µL of a liquid MB 1X culture of Pseudovibrio denitrificans(dated 12th August) in a part and with 5µL of a liquid M9 1X culture Pseudovibrio denitrificans (dated 12th August) in the other part.
Pre-cultures
Cultures in MB 1X and M9 1X of Pseudovibrio were passed by a 1/10 dilution and let incubate overnight at 30°C. New cultures of different E.Coli were also started from glycerol or plate and let incubated overnight at 37°C.
From glycerol stocks:
From plates:
Bacteria tranformed with plasmid were cultivated with the corresponding antibiotic for the selection. For each medium we make a negative contrôle without bacteria. Aug 16
