The preparation and recombination of the protons of the receptor cells.

1. Master the principles and methods ₂ using CaCl 2 to prepare receptor cells.

2. Learn and master the screening methods for the transformation and recombination of prosury DNA.

" It is a common phenomenon in the world ofmicrobes, in which a bacterial line changes its genetic traits by absorbing the granule DNA of another bacterial line, a phenomenon called transformation, in which cells that obtain external DNA are called transformers.

In gene cloning technology, the so >-called transformation refers to the process by which prosurfic or recombined prosurfic particles are imported into the subject cells, expressing the corresponding selection marker genes, and developing the converters on selectiveunder certain conditions of ""> culture".

the prosurges must be converted into bacterial cells for amplification and expression, resulting in a large number of cloned genes that allow us to perform further DNA operations, such as subclonals, or obtain their expression products. The conversion efficiency is related to the physiological state of the subject bacteria. The state in which bacteria absorb external DNA is at its highest is called receptor cells.

Some types of bacteria are in a receptor state at any stage of their growth, while others are only in a receptor state when they are in a certain period of growth (generally early and medium-term development of the number of pairs), such as E. coli used in this experiment. Treating bacteria with a certain concentration of CaCl2 in the early and middle stages of the growth of the number can greatly improve the ability of bacteria to absorb DNA molecules in their surroundings.

explanation for this phenomenon is that CaCl2 increases the permeability of bacterial cell walls, thereby increasing conversion rates. This conversion method is called "chemical method". At present, electric shock can also be used, through an instantaneous high-voltage current, the formation of holes in the cell, so that the external DNA into the cell, so as to achieve cell transformation.

electrocution conversion is often 1 to 2 orders of magnitude more efficient than chemical methods, reaching 1 x 108 transformers/g DNA, or even 1 x 109 transformers/g DNA, so it is often used for conversion or genetic screening when building libraries.

microbial transformation is a common technique of genetic engineering, E. coli is the most commonly used subject bacteria in genetic engineering, this experiment is the use of the previous experiments obtained by the recombinative proton transformation of E. coli cells.

1, preparation of receptor cells (aseptic operation)

2, the transformation of receptor cells

(1) When reversing the plate should avoid the culture base temperature is too high, if the temperature is too high, the added ampicillin will fail, and the culture base solidified after the surface and lid will form a large amount of condensate, easy to cause pollution and affect the formation of monobacteria. If you feel the culture base with the palm of your hand and feel it is hot but tolerable, the culture base temperature is about 55 degrees C. The prepared LB/Amp tablet can be stored in a refrigerator at 4 degrees C for 2 months, and over time ampicillin will fail. The LB/Amp tablet with IPTG/X-Gal is best used now.