Lab Experiment 37.2 : Preparation of E.coli competent cells for Electroporation.
Background Information: The permeability of the cell membrane can be increased in the presence of high external electric field. This approach is used to deliver the molecular probe, drugs or DNA into the different types of cells. Several hundred volts across plasma membrane is applied in this process. The deliver process is ~10 times more efficient than calcium chloride mediated transformation. Electroporation is performed with the help of electroporator, which can create very high electric field in a specialized cuvette (Figure 37.2).
Figure 37.2:Equipments required for electroporation. (A) electroporator (B) Cuvette.
Material:
1. DH5α Host cells stock
2. Triple distilled water.
Preparation of Competent Cell- The whole process of E.coli competent cells preparation is as follows:
1. Inoculate single colony into the 100ml LB media and allow the cells to grow at 370C, 180rpm until
OD600 nm reaches to the 0.4-0.6.
2. Centrifuge the bacterial culture at 4000 rpm, at 4°C 10 min. Discard the supernatant.
3. Resuspend the cell pellet gently in 5-10ml triple distilled water (multiple times).
4. Centrifuge the bacterial suspension at 4000 rpm, at 4°C 10 min. Discard the supernatant.
5. Resuspend cells gently in 3.0 ml of ice-cold distilled water.
6. Centrifuge the bacterial suspension at 4000 rpm, at 4°C 10 min. Discard the supernatant.
7. Resuspend cells gently in 3.0 ml of ice-cold triple distilled water containing 10% glycerol and store in small aliquot (100µl) at -800C. The cells can be used for transformation (discussed in later lectures).