Difference between revisions of "Team:Aix-Marseille/Protocols"
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<h2 class="title wow Hinge"><span style="color:#8E3B8C">Protocols</h2></span> | <h2 class="title wow Hinge"><span style="color:#8E3B8C">Protocols</h2></span> | ||
<div class="space30"></div> | <div class="space30"></div> | ||
| − | <p | + | <p class="space20"><div align="justify">Transformation</div></p> |
| − | + | <p> Plasmids transformation : | |
| + | |||
| + | Add 20 ng of plasmid to 100 µL of competent cells thawed in ice | ||
| + | |||
| + | Incubate 30-45 min in ice | ||
| + | |||
| + | Thermal shock : put tubes in the Thermomixer at 42°C during 2 min | ||
| + | |||
| + | Incubate 5 min in ice | ||
| + | |||
| + | Add 900 µL of LB | ||
| + | |||
| + | Incubate 1 hour at 37°C with agitation | ||
| + | |||
| + | Spread 100 µL on LB limp (with antibiotic) | ||
| + | |||
| + | To make negative control, follow the same procedure but without add plasmids and spread 300 µL | ||
| + | |||
| + | Ligation transformation: | ||
| + | |||
| + | Add 20 ng of ligation’s product to 100 µL of competent cells thawed in ice | ||
| + | |||
| + | Incubate 30-45 min in ice | ||
| + | |||
| + | Thermal shock : put tubes in the Thermomixer at 42°C during 2 min | ||
| + | |||
| + | Incubate 5 min in ice | ||
| + | |||
| + | Add 900 µL of LB | ||
| + | |||
| + | Incubate 1 hour at 37°C with agitation | ||
| + | |||
| + | Centrifuge 5 min at 5000 rpm | ||
| + | |||
| + | Eliminate 850 µL of medium | ||
| + | |||
| + | Suspend the pellet | ||
| + | |||
| + | Spread 150 µL on LB limp (with antibiotic) | ||
| + | To make negative control, follow the same procedure but without add plasmids and spread 300 µL <p/> | ||
</div> | </div> | ||
Revision as of 17:15, 15 September 2015
Protocols
Plasmids transformation : Add 20 ng of plasmid to 100 µL of competent cells thawed in ice Incubate 30-45 min in ice Thermal shock : put tubes in the Thermomixer at 42°C during 2 min Incubate 5 min in ice Add 900 µL of LB Incubate 1 hour at 37°C with agitation Spread 100 µL on LB limp (with antibiotic) To make negative control, follow the same procedure but without add plasmids and spread 300 µL Ligation transformation: Add 20 ng of ligation’s product to 100 µL of competent cells thawed in ice Incubate 30-45 min in ice Thermal shock : put tubes in the Thermomixer at 42°C during 2 min Incubate 5 min in ice Add 900 µL of LB Incubate 1 hour at 37°C with agitation Centrifuge 5 min at 5000 rpm Eliminate 850 µL of medium Suspend the pellet Spread 150 µL on LB limp (with antibiotic) To make negative control, follow the same procedure but without add plasmids and spread 300 µL
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