*Preparation of competent cell-:


Natural competence was first discovered by Frederich Griffith in 1928.
*Principle:-
Competent cells are ready to use bacterial cells that possess more easily altered cell walls by which  foreign DNA can be passed through easily. Most types of cells cannot take up DNA efficiently unless they have been exposed to special chemical or electrical treatments to make them competent . However, some types of bacteria are naturally transformable, which means they can take up DNA from their environment without requiring special treatment. The exact mechanisms involved in artificial competence are not yet known well. ln CaCl2 method, the competency can be obtained by creating pores in bacterial cells by suspending them in a solution containing high concentration of calcium.

Artificial competence is not encoded in the cell's genes. Instead it is a laboratory procedure by which cells are  made permeable to DNA, with  conditions that do not normally occur in nature. This procedure is comparatively easy and simple, and can be used in the genetic engineering of bacteria but in general transformation efficiency is low.  Methods for preparing the competent cells derive from the work of Mandel and Higa who developed a simple treatment based on soaking the cells in cold CaCl2 . There are two main methods for the preparation of competent cells .  1)Calcium chloride method:-
     ln CaCl2 method, the competency can be obtained by creating pores in bacterial cells by suspending them in a solution containing high concentration of calcium.

 2) Electroporation:-
The standard method for making the bacteria permeable to DNA involves treatment with calcium ions. Brief exposure of cells to an electric field also allows the bacteria to take up DNA and this process is called as electroporation . 


*Protocol 
CaCl2 method--

Inoculate a 5 ml overnight-culture in LB-medium without antibiotics (for DH5a) or with appropriate antibiotics (for other strains).
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Grow over night at 37 ˚C at 250 rpm.
Luria-Bertani (LB) broth, makes 1L
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Inoculate 100 ml LB-medium with 1/100 volume of the overnight culture and incubate to an OD600 = 0.5 - 0.6 at 37 °C and 230 rpm.
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Transfer cells to two sterile 50 ml falcon tubes and incubate on ice for 20 min.
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From this step on, always keep cells cold
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Centrifuge for 5 min at 4500 rpm and 4 °C (pre-cool centrifuge) and discard the supernatant.
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Carefully suspend each pellet in 20 ml of ice-cold 100 mM CaCl2 and incubate on ice for 1 h.
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Centrifuge for 5 min at 4500 rpm and 4 °C (pre-cool centrifuge) and discard the supernatant.
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Carefully suspend each pellet in 2 ml icecold 85 mM CaCl2 containing 15% Glycerol.
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Pipette 50 - 200 µl aliquots in prepared, pre-cooled 1.5 ml tubes (work on ice), shock-freeze in liquid nitrogen or on dry ice and store at -80 °C.

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