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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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| - | <p>We started to make 200 mL of BL21 and DH5α E. coli strains and 400 mL of TOP10.<br/></p> | + | <p>We started to make 200 mL of BL21 and DH5α E. coli strains and 400 mL of TOP10 to make competent cells.<br/></p> |
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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> | ||
Revision as of 16:08, 28 July 2013
Week 1: 17th June - 23rd June
Tuesday, 19th June
As a start for the labwork, we first decided to make competent cells. 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
In a 15 mL tube, add 2 mL of LB medium and inoculate cells from glycerol
Thursday, 20th June
We started to make 200 mL of BL21 and DH5α E. coli strains and 400 mL of TOP10 to make 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
To check the quality of our work, each strain has been plated on LB medium with ampicillin, kanamycin or chloramphenicol in order to evaluate if it contaminated or not. Furthermore, to evaluate wether our strains are competent or not, we also transformed our bacteria with a pSB1A3 plasmid (red colonies) and plated them on LB medium with ampicillin only.
For 100 µL of chemical competent cells, add 1 µL of plasmidic DNA.
Let 30 minutes on ice.
Let the cells at 42°C for exactly 50 seconds.
Let 5 minutes on ice.
Resuspend the cells with 1 mL of LB spread on a petri dish then let it at 37°C.
