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    • Last Update: 2020-11-03
    • Source: Internet
    • Author: User
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    transformation is the key to external
    molecules are introduced into the subject cells as a means of obtaining new genetic characteristics, which are basic experimental techniques in the fields of
    engineering, etc.The
    transformation process uses the subject cells that are typically variants that limit the modification system defects, i.e. mutants that do not contain restrictive endoenzymes and methylases (R, M), which can tolerate the entry of exogenetic DNA molecules into the body and be steadily passed on to future generations. After the treatment of the subject cells by special methods such as electros shock, CaCl2, rbCl (KCl) and other
    chemical reagents
    ), the permeability of the cell membrane changes temporarily, becoming a permeable cell that allows exogenous DNA molecules to enter the compenent cells. DNA molecules entering the subject cells are transferred through replication and expression, resulting in new genetic features in the subject cells. The transformed cells are cultured in a
    base and the converters (Transformant, a subject cell with heterogenetic DNA molecules) can be screened. At present, the commonly used methods of percepto-state cell preparation are CaCl2 and RbCl (KCl) method, RbCl (KCl) legal preparation of the percepto-state cell conversion efficiency is high, but CaCl2 method is simple and easy to do, and its conversion efficiency can fully meet the requirements of general experiments, the preparation of the percepto-state cells temporarily not used, can be added to the total volume of 15% of the sterile glycemic glycemic oil in -70 degrees C preservation (half a year), so CaCl2 method for wider use.
    in order to improve conversion efficiency, the following important factors should be considered in the experiment:
    1. Cell growth status and density: Do not use cultured bacteria that have been transferred multiple times or stored at 4 degrees C, preferably directly transferred from the species preserved in -70 degrees C or -20 degrees C glycera for the preparation of receptor cells. Cell growth density is good for just entering the number of years, which can be controlled by monitoring the OD600 of the culture fluid. At 0.5 for the OD600 of the DH5 alpha strain, the cell density is around 5×107 /ml (different strains vary), which is appropriate. High or insufficient density can affect conversion efficiency.
    2. Mass and concentration of the prosulte: the prospolyte DNA used for transformation should be primarily ultra-helical DNA (cc
    ). The conversion efficiency is directly related to the concentration of the external DNA within a certain range, but the conversion efficiency is reduced when the amount of the added exonyn DNA is too large or the volume is too large. 1 ng of cccDNA can saturate 50 sl of the resurgency cells. In general, DNA solutions should not exceed 5% of the volume of receptor cells.
    Quality of reagents
    : Reagents, such as CaCl2, are required to be of the highest purity (GR. or AR.) and are made of ultra-pure water, preferably in the cold and dark of

    4. Prevent contamination of clutter and miscellaneous DNA: the entire operation should be carried out under sterile conditions, the utensils used, such as centrifugal tubes, tip first class is preferably new, and by autoculsive sterilization treatment, all reagents should be sterilized, and attention should be taken to prevent contamination by other reagents, DNA enzymes or miscellaneous DNA, otherwise it will affect conversion efficiency or the transfer of hybrid DNA, for future screening, identification bring unnecessary trouble.
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