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Team:UCL/protocols
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<h1>Protocols</h1> | <h1>Protocols</h1> | ||
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| + | <h4>Creating Competent Cells</h4> | ||
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| + | <p>Materials | ||
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| + | LB | ||
| + | Falcon Tubes | ||
| + | Ice | ||
| + | Chilled centrifuge | ||
| + | CaCL | ||
| + | Microtubes (300ul/tube) | ||
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| + | Procedure | ||
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| + | Inoculate a single colony into 5ml Lb in 50ml falcon tube. Grown O/N @ 37oC | ||
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| + | Use 1ml to inoculate 100ml of LB in 250ml bottle the next morning. | ||
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| + | Shake @ 37oC for 1.5-3 hours. | ||
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| + | Or | ||
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| + | Inoculate a single colony into 25ml LB in a 250ml bottle in the morning | ||
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| + | Shake @ 37oC for 4-6 hours. | ||
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| + | Then… | ||
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| + | Put the cells on ice for 10mins (keep cold from now on). | ||
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| + | Collect the cells by centrifugation in the big centrifuge for 3 minutes @ 6Krpm. | ||
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| + | Decant supernatant and gently resuspend on 10ml cold 0.1M CaCl (cells sensitive to mechanical disruption). | ||
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| + | Incubate on ice x 20 minutes | ||
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| + | Centrifuge as in 2. | ||
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| + | Discard supernatant and gently resuspend on 5ml cold 0.1M CaCl/15%Glycerol. | ||
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| + | Dispense in microtubes (300ųl/tube). Freeze at -80oC. | ||
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| + | </p> | ||
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</div> | </div> | ||
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Revision as of 14:30, 5 September 2014
