Team:Evry/Notebook/w1

From 2013.igem.org

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<h2>Thursday, 20th June</h2>
<h2>Thursday, 20th June</h2>
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We started to make 200 mL of BL21 and DH5α E. coli strains and 400 mL of TOP10.
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<b><div align="center">Protocol for competent cells</b><br/></div>
<b><div align="center">Protocol for competent cells</b><br/></div>
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Inoculation of the bacteria:<br/>
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For 200 ml LB medium, add 400 µL of strain sample.<br/>
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Let the bacteria grow until it reaches an OD between 0,3 and 0,35.<br/>
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Once it reached the right OD, put the medium on ice for 30 minutes to slow down growth.<br/>
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Split the 200 mL into 4x50 mL tubes then centrifuge at 3000 rpm for 5 minutes at 4°C and suppress supernatant afterwards.<br/>
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Resuspend the cells with 5 mL of Cacl2 at 0,1M for each 50 mL tube.<br/>
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Again, put the medium on ice for 30 minutes.<br/>
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Centrifuge at 3000 rpm for 5 minutes at 4°C then suppress supernatant.<br/>
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Resuspend the cells with 1 mL of Cacl2 at 0,1M + 15% glycerol for each 50 mL tube.<br/>
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Split the total 4 mL into 40 tubes containing each 100 µL of concentrated cell solution. This step should be executed fast enough and on ice.<br/>
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For 200 ml LB medium, add 400 µL of strain sample (BL21 or DH5α).<br/>
 
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However, for the TOP10 strain, we decided to make 400 mL LB medium with 800 µL of strain sample. In fact, TOP10 is a cloning strain and we will use it in our initial steps.
 
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Revision as of 15:25, 28 July 2013

Iron coli project

Week 1: 17th June - 23rd June

Tuesday, 19th June

As a start for the labwork, we first decided to make competent cells. As a consequence, we grew from glycerol samples three E. coli strains in 2 mL of LB medium.
The chemically competent strains chosen are as follows:

BL21
DH5α
TOP10

Thursday, 20th June


We started to make 200 mL of BL21 and DH5α E. coli strains and 400 mL of TOP10.
Protocol for competent cells

For 200 ml LB medium, add 400 µL of strain sample.
Let the bacteria grow until it reaches an OD between 0,3 and 0,35.
Once it reached the right OD, put the medium on ice for 30 minutes to slow down growth.
Split the 200 mL into 4x50 mL tubes then centrifuge at 3000 rpm for 5 minutes at 4°C and suppress supernatant afterwards.
Resuspend the cells with 5 mL of Cacl2 at 0,1M for each 50 mL tube.
Again, put the medium on ice for 30 minutes.
Centrifuge at 3000 rpm for 5 minutes at 4°C then suppress supernatant.
Resuspend the cells with 1 mL of Cacl2 at 0,1M + 15% glycerol for each 50 mL tube.
Split the total 4 mL into 40 tubes containing each 100 µL of concentrated cell solution. This step should be executed fast enough and on ice.


Friday, 21st June

The tree following E. coli strains have been prepared to be chemically competent: BL21, DH5α and TOP10.


To check the quality of our work as well as their competence, each strain has been plated on LB medium with ampicillin, kanamycin or chloramphenicol. We also transformed our bacteria with a random plasmid and plated them on LB medium with ampicillin only. We incubated our petri dishes for the week end at 30°c