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Team:Grenoble/Biology/Protocols/Transformation 2
From 2012.igem.org
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<li>Let sit for 30 minutes on ice.</li> | <li>Let sit for 30 minutes on ice.</li> | ||
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<li>Prepare the heating block for a temperature of 42°C.</li> | <li>Prepare the heating block for a temperature of 42°C.</li> | ||
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<li>Incubate cells on ice for 2 min.</li> | <li>Incubate cells on ice for 2 min.</li> | ||
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<li>Add 1 mL of LB medium at room temperature.</li> | <li>Add 1 mL of LB medium at room temperature.</li> | ||
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<li>Incubate for 1 hour at 37°C on shaker.</li> | <li>Incubate for 1 hour at 37°C on shaker.</li> | ||
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<li>Spread 100-300 µL onto a plate made with appropriate antibiotic.</li> | <li>Spread 100-300 µL onto a plate made with appropriate antibiotic.</li> | ||
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Revision as of 19:30, 27 August 2012
Transformation of E. coli competent cells (heat shock)
Goal
Transform E. coli competent cells whith foreign DNA.Protocol
Following are the instructions from the OpenWetWare website.- Prepare ice backs.
- 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.
- Prepare the heating block for a temperature of 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 made with appropriate antibiotic.
- Grow overnight at 37°C.
SAFETY AND USEFUL RECOMMANDATION |
|---|
In order to reduce the risk of ice that could fall, it
is recommended to place the back near the place manipulations and in case of any move,
it is better to take eppendorfes in an eppendorf rack. That is why it is important to
know where manipulations will be done. |
SAFETY AND USEFUL RECOMMANDATION |
|---|
When doing that it is necessary to wear special gloves
which protect against frostbite. Indeed cells are in a freezer where the temperature
is around -80°C. Moreover the ice melts, therefore it is also important to be careful
to not place ice near electric devices. There is a risk of electrocution. To use cells
the substance must have melt. |
SAFETY AND USEFUL RECOMMANDATION |
|---|
During this step, cells are manipulated. It is then necessary
to work in a sterile spot, by using a flow hood. |
SAFETY AND USEFUL RECOMMANDATION |
|---|
It is important to know before manipulating how the device has to
be used. The heating block takes à long time before reaching the good temperature. Therefore
it is recommended to ask the device to reach a temperature higher than the one that has to be
reached for the manipulation. Asking a temperature of 53°C at the beginning accelerates the
increase of the temperature. But after that the temperature must be reduce to 42°C. The
temperature here is not very high. But in case of manipulation asking for higher temperature,
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 used anymore. |
SAFETY AND USEFUL RECOMMANDATION |
|---|
Users must pay attention that eppendorfs are well closed. |
SAFETY AND USEFUL RECOMMANDATION |
|---|
This has to be done in a sterile spot. |
SAFETY AND USEFUL RECOMMANDATION |
|---|
This has to be done in a sterile spot. |
SAFETY AND USEFUL RECOMMANDATION |
|---|
When leaving things in the lab that keep on working, users
must tell the person in charge of the lab and placing an inscription on the device in
order to not have someone who turns the device off during the absence of the other manipulators.
|
