Team:Evry/Notebook/w2

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<h1>Week 2: 24th June - 30th June</h1>
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<h1>Week 2: 24<sup>th</sup> June - 30<sup>th</sup> June</h1>
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<h2> Monday, June 24th </h2>
<h2> Monday, June 24th </h2>
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Competents cells made on friday 21st was plated and incubated the whole week-end at 30°C. Today we analyzed results and it seems that medium had been contaminated.
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<p> 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.<br/>
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<b><div align="center">Protocol for LB medium preparation</b><br/></div>
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<br/>New competents cells will be made.
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Add 20 g of LB broth into 1000 mL of desalted water and autoclave.<br/>
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<b><div align="center">Protocol for antibiotic solutions preparation</b><br/></div>
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<p>Kanamycin is used as an effective concentration of 10-50 µg/ml and at a storage concentration of 10 mg/ml<br/>
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Carbenicillin is used as an effective concentration of 20-60 µg/ml and at a storage concentration of 50 mg/ml<br/>
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Chloramphenicol is used as an effective concentration of 25-170 µg/ml and at a storage concentration of 34 mg/ml (ethanol)<br/>
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Tetracyclin is used as an effective concentration of 10-50 and at a storage concentration of 5 mg/ml (ethanol)<br/></p>
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<h2>Tuesday, June 25th</h2>
<h2>Tuesday, June 25th</h2>
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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.
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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 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.</p>
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<h2>Wednesday, June 26th</h2>
<h2>Wednesday, June 26th</h2>
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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.
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<p>The results of the competent cells protocol is as follows:<br/>
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DH5α strain was not contaminated but competent<br/>
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TOP10 strain was contaminated only on the Kanamycin plate but competent<br/>
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BL21 strain was completely contaminated and, thus, not usable.<br/></p>
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<h2> Thurdsay, June 27th </h2>
<h2> Thurdsay, June 27th </h2>
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We designed the plasmid constructions and primers we will use.
 
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<p>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.</p>
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<h2> Friday, June 28th </h2>
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<p>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.</p>
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{{:Team:Evry/foot}}

Latest revision as of 13:56, 25 August 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 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.