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Team:UCL/protocols
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<h4>Creating Competent Cells</h4> | <h4>Creating Competent Cells</h4> | ||
| - | <p>Materials | + | <p><b>Materials</b><br/> |
| - | LB | + | LB<br/> |
| - | Falcon Tubes | + | Falcon Tubes<br/> |
| - | Ice | + | Ice<br/> |
| - | Chilled centrifuge | + | Chilled centrifuge <br/> |
| - | CaCL | + | CaCL<br/> |
| - | Microtubes (300ul/tube) | + | Microtubes (300ul/tube)<br/> |
| - | Procedure | + | <b>Procedure</b><br/> |
| + | Inoculate a single colony into 5ml Lb in 50ml falcon tube. Grown O/N @ 37oC<br/> | ||
| + | Use 1ml to inoculate 100ml of LB in 250ml bottle the next morning.<br/> | ||
| + | Shake @ 37oC for 1.5-3 hours.<br/> | ||
| - | + | Or<br/> | |
| - | + | Inoculate a single colony into 25ml LB in a 250ml bottle in the morning<br/> | |
| + | Shake @ 37oC for 4-6 hours.<br/> | ||
| - | + | Then…<br/> | |
| - | + | Put the cells on ice for 10mins (keep cold from now on).<br/> | |
| - | + | Collect the cells by centrifugation in the big centrifuge for 3 minutes @ 6Krpm.<br/> | |
| - | + | Decant supernatant and gently resuspend on 10ml cold 0.1M CaCl (cells sensitive to mechanical disruption).<br/> | |
| - | + | Incubate on ice x 20 minutes<br/> | |
| - | + | Centrifuge as in 2.<br/> | |
| - | + | Discard supernatant and gently resuspend on 5ml cold 0.1M CaCl/15%Glycerol.<br/> | |
| - | + | Dispense in microtubes (300ųl/tube). Freeze at -80oC.<br/> | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | Put the cells on ice for 10mins (keep cold from now on). | + | |
| - | + | ||
| - | Collect the cells by centrifugation in the big centrifuge for 3 minutes @ 6Krpm. | + | |
| - | + | ||
| - | Decant supernatant and gently resuspend on 10ml cold 0.1M CaCl (cells sensitive to mechanical disruption). | + | |
| - | + | ||
| - | Incubate on ice x 20 minutes | + | |
| - | + | ||
| - | Centrifuge as in 2. | + | |
| - | + | ||
| - | Discard supernatant and gently resuspend on 5ml cold 0.1M CaCl/15%Glycerol. | + | |
| - | + | ||
| - | Dispense in microtubes (300ųl/tube). Freeze at -80oC. | + | |
</p> | </p> | ||
Revision as of 14:31, 5 September 2014
