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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 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 contamination.
Wednesday, June 26th
The results of the competent cells protocol is as follows:
DH5α strain was not contaminated but competent
TOP10 strain was contaminated only on the Kanamycin plate but competent
BL21 strain was completely contaminated and, thus, not usable.
Thurdsay, June 27th
We finished designing are first two plasmids and constructed the primers to extract the natural promoters from the genomic DNA from E. coli and the different parts for Golden Gate assembly.
Friday, June 28th
We want to obtain reasonnable competent cells for our oncoming transformation for newt week. Thus, we decided to plate the three strains on LB-Agar medium in order to isolate one and only one colony for monday when we'll start over the whole competent process.
