Transformation of E. coli competent cells (heat shock)

Goal

Protocol

1. Prepare ice backs.



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.



2. Thaw TSS E. coli competent cells (from the freezer) on ice.



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.



3. Add DNA, pipette gently to mix (1 µL of prepped plasmid is more than enough).



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.



4. Let sit for 30 minutes on ice.
5. Prepare the heating block for a temperature of 42°C.



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.



6. Incubate cells for 30 seconds at 42°C.



SAFETY AND USEFUL RECOMMANDATION
Users must pay attention that eppendorfs are well closed.



7. Incubate cells on ice for 2 min.
8. Add 1 mL of LB medium at room temperature.



SAFETY AND USEFUL RECOMMANDATION
This has to be done in a sterile spot.



9. Incubate for 1 hour at 37°C on shaker.
10. Spread 100-300 µL onto a plate made with appropriate antibiotic.



SAFETY AND USEFUL RECOMMANDATION
This has to be done in a sterile spot.



11. Grow overnight at 37°C.



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.