Team:Evry/Protocols/01

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<h1 align='center'> Competents cells </h1>
<h1 align='center'> Competents cells </h1>
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First prepare the following solutions required to make competent cells:
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Solution for 1M Cacl2:
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Add 14,30g of CaCl2 into 100 ml desalted water
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Solution for 0,1M Cacl2:
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Add 50 mL of CaCl2 1M solution into 450 ml of desalted water
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Solution for 0,1M Cacl2 + 15% glycerol:
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Add 50 mL of CaCl2 1M solution and 75 mL of glycerol 100% into 450 ml of desalted water
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For 200 ml LB medium, add 400 µL of strain sample.
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Let the bacteria grow until it reaches an OD between 0,3 and 0,35.
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Once it reached the right OD, put the medium on ice for 30 minutes to slow down growth.
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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.
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Resuspend the cells with 5 mL of Cacl2 at 0,1M for each 50 mL tube.
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Again, put the medium on ice for 30 minutes.
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Centrifuge at 3000 rpm for 5 minutes at 4°C then suppress supernatant.
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Resuspend the cells with 1 mL of Cacl2 at 0,1M + 15% glycerol for each 50 mL tube.
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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.
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Revision as of 14:45, 29 July 2013

Competents cells

First prepare the following solutions required to make competent cells: Solution for 1M Cacl2: Add 14,30g of CaCl2 into 100 ml desalted water Solution for 0,1M Cacl2: Add 50 mL of CaCl2 1M solution into 450 ml of desalted water Solution for 0,1M Cacl2 + 15% glycerol: Add 50 mL of CaCl2 1M solution and 75 mL of glycerol 100% into 450 ml of desalted water 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.

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