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Team:Evry/Notebook/w2
From 2013.igem.org
Week 2: 24th June - 30th June
Monday, June 24th
Our competents cells made on friday the 21st were plated and incubated the whole week-end at 30°C. Today, we analyzed the plates and came to the conclusion that the medium was contaminated due to poor protocol execution. As a consequence, we decided to make new LB medium and recalculate the antibiotic concentrations.
Add 20 g of LB broth into 1000 mL of desalted water and autoclave.
Kanamycin is used as an effective concentration of 10-50 µg/ml and at a storage concentration of 10 mg/ml
Carbenicillin is used as an effective concentration of 20-60 µg/ml and at a storage concentration of 50 mg/ml
Chloramphenicol is used as an effective concentration of 25-170 µg/ml and at a storage concentration of 34 mg/ml (ethanol)
Tetracyclin is used as an effective concentration of 10-50 and at a storage concentration of 5 mg/ml (ethanol)
Tuesday, June 25th
New competent cells have been made with the same bacterial strains (BL21, DH5α and TOP10). We thought that the contamination of our cells was due to either the CaCl2 solution that may not have been sterile and/or incorrect manipulated. As a consequence, we prepared new CaCl2 solutions made sure to autoclave them before usage. We ran the same tests as monday the 24th to evaluate the quality of our work and check for potential contaminatione.
Wednesday, June 26th
The DH5α strain was not contaminated and competent. The TOP10 strain was contaminated only on the Kanamycin petri dish and competent. The BL21 strain was contaminated and not usable.
Thurdsay, June 27th
We designed the plasmid constructions and primers we will use.
