Difference between revisions of "Team:Aix-Marseille/Protocols"
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Add 20 ng of plasmid to 100 µL of competent cells thawed in ice<br /> | Add 20 ng of plasmid to 100 µL of competent cells thawed in ice<br /> | ||
| − | |||
Incubate 30-45 min in ice<br /> | Incubate 30-45 min in ice<br /> | ||
| − | |||
Thermal shock : put tubes in the Thermomixer at 42°C during 2 min<br /> | Thermal shock : put tubes in the Thermomixer at 42°C during 2 min<br /> | ||
| − | |||
Incubate 5 min in ice<br /> | Incubate 5 min in ice<br /> | ||
| − | |||
Add 900 µL of LB <br /> | Add 900 µL of LB <br /> | ||
| − | |||
Incubate 1 hour at 37°C with agitation<br /> | Incubate 1 hour at 37°C with agitation<br /> | ||
| − | |||
Spread 100 µL on LB limp (with antibiotic)<br /> | Spread 100 µL on LB limp (with antibiotic)<br /> | ||
| − | |||
To make negative control, follow the same procedure but without add plasmids and spread 300 µL<br /> | To make negative control, follow the same procedure but without add plasmids and spread 300 µL<br /> | ||
| − | |||
Ligation transformation:<br /> | Ligation transformation:<br /> | ||
| − | |||
Add 20 ng of ligation’s product to 100 µL of competent cells thawed in ice<br /> | Add 20 ng of ligation’s product to 100 µL of competent cells thawed in ice<br /> | ||
| − | |||
Incubate 30-45 min in ice<br /> | Incubate 30-45 min in ice<br /> | ||
| − | |||
Thermal shock : put tubes in the Thermomixer at 42°C during 2 min<br /> | Thermal shock : put tubes in the Thermomixer at 42°C during 2 min<br /> | ||
| − | |||
Incubate 5 min in ice<br /> | Incubate 5 min in ice<br /> | ||
| − | |||
Add 900 µL of LB <br /> | Add 900 µL of LB <br /> | ||
| − | |||
Incubate 1 hour at 37°C with agitation<br /> | Incubate 1 hour at 37°C with agitation<br /> | ||
| − | |||
Centrifuge 5 min at 5000 rpm<br /> | Centrifuge 5 min at 5000 rpm<br /> | ||
| − | |||
Eliminate 850 µL of medium <br /> | Eliminate 850 µL of medium <br /> | ||
| − | |||
Suspend the pellet <br /> | Suspend the pellet <br /> | ||
| − | |||
Spread 150 µL on LB limp (with antibiotic)<br /> | Spread 150 µL on LB limp (with antibiotic)<br /> | ||
To make negative control, follow the same procedure but without add plasmids and spread 300 µL <p/> | To make negative control, follow the same procedure but without add plasmids and spread 300 µL <p/> | ||
Revision as of 17:18, 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
TITTLE
