Team:Evry/Notebook/w2

From 2013.igem.org

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<p>New competent cells have been made with the same strains of bacteria. We thought that the contamination of our cells was due either to the solution of CaCl2 that we used which may had not been sterile and/or incorrect manipulations. This time, we prepared new CaCl2 solutions that we made sure to autoclave correctly. Then, same tests as we did before of contamination and competence have been made.</p>
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<p>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.</p>
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<p>The DH5a 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.</p>
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<p>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.</p>

Revision as of 16:38, 28 July 2013

Iron coli project

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.


Protocol for LB medium preparation

Add 20 g of LB broth into 1000 mL of desalted water and autoclave.


Protocol for antibiotic solutions preparation

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.

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