Team:Grenoble/Biology/Protocols/Transformation 2
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Transformation of E. coli competent cells (heat shock)
Goal
Transform E. coli competent cells with foreign DNA.Protocol
Following are the instructions from the OpenWetWare website.- Prepare ice containers.
- Thaw TSS E. coli competent cells (from the freezer) on ice.
- Add DNA, pipette gently to mix (1 µL of prepped plasmid is more than enough).
- Let sit for 30 minutes on ice.
- Set the heating block at 42°C.
- Incubate cells for 30 seconds at 42°C.
- Incubate cells on ice for 2 min.
- Add 1 mL of LB medium at room temperature.
- Incubate for 1 hour at 37°C on shaker.
- Spread 100-300 µL onto a plate complemented with appropriate antibiotic.
- Grow overnight at 37°C.
To prevent the ice container from falling off the workbench, it is
recommended to place it away from edges. In addition, it is better to put eppendorfs in a special rack.
It is important to know where manipulations will be achieved in order to apprehend the possible risks. |
Using the -80°C freezer requires to wear special gloves
which protect against frostbite. Moreover ice melts, therefore it is also important to be careful
not to place the iced object near electric devices. There is a risk of electrocution. |
During this step, cells are manipulated. It is thus necessary
to work in the sterile environment provided by the flow hood. |
It is important to know how a device works before using it.
The heating block takes a long time before reaching a defined temperature. Therefore,
it is recommended to switch it on a while before you need it. It is possible to accelerate the temperature increase by setting the device to
a higher temperature than the one required. Of course it has to be set to 42∞C. In case the protocol requires a higher, the
users must be careful of the electric cables. They must not touch the hot spot of the device.
Users must also not forget to stop the device once it is not needed any further. |
Make sure that the eppendorfs are closed. |
This has to be done in a sterile area. |
This has to be done in a sterile environment. |
When leaving devices on working devices in the laboratory, users
must tell the person in charge of the laboratory. In addition, they must place a note on the device in
order to inform the personnal of the laboratory that an experiment is in progress.
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