Methods of cell lysis include chemical lysis, enzymatic cleavage, and mechanical lysis. Chemical lysis and enzymatic cleavage are generally mild methods and usually do not break much of the DNA. The methods commonly used to extract and purify DNA when using these two methods (including SDS and lysozyme treatment). Mechanical lysis can uniformly lyse cells, and mechanical treatment has higher and stronger cleavage efficiency and lower selectivity than chemical cleavage. Mechanical treatment can lyse cells more vigorously and comprehensively, but can cause DNA breakage.

First, the mechanical cracking method mainly has the following two

1. Thermal shock, which is a repeated mechanical method of mechanical lysis, usually consists of freezing and thawing. Principle: The cell structure is broken due to the formation of ice particles in the cells and the increase in the salt concentration of the remaining cell fluid. Freezing is usually carried out on liquid nitrogen or -20 ° C ice, and thawing can be carried out in a 37, 50, 65 or 100 ° C water bath. Heat shock is milder than chemical lysis, but it is very effective. There are data showing that 90% cell lysis rate is obtained by heat shock and lysozyme and SDS.

2. Ultrasonication uses both ultrasonic heating methods to break up cells. However, this treatment will lead to DNA breakage, so the heating should not be too intense. The ultrasonic time and gap time should be set. Generally, the ultrasonic time is less than 5 seconds, and the gap time is better than the ultrasonic time. These are beneficial to protect the enzyme activity. . Bead-beating is also a commonly used mechanical treatment. It has been reported that bead-beating is better than heat shock and chemical lysis. Although the DNA yield is higher, the DNA fragments usually obtained are smaller.

Second, chemical lysis and enzymatic cleavage (combined in the extraction of nucleic acids)

Mainly lysate treatment, the main purpose of cell lysate is the following: (1) use of detergent to destroy lipid bilayer, rupture cells; (2) dissolved protein; (3) protein denaturation to make it stable; (4) inhibition of protease activity.

The composition of the lysate differs mainly for different purposes, mainly in the extraction of nucleic acids and proteins. When extracting RNA or DNA, we mainly want to fully lyse the cells to get more nucleic acids; if our purpose is protein, then we should consider the lysate according to the location and characteristics of the protein, after extracting the protein, and then according to the experiment. Refolding protein is required. The following are commonly used reagents for cell lysis and their effects: 50 mM Tris-HCl pH 7.4 (buffer system), 150 mM NaCl (isotonic system), 1 mM PMSF (strong protease inhibitor), 1 mM EDTA (denaturing agent and Stabilizer), 5 μg/ml Aprotinin (protease inhibitor), 5 μg/ml Leupeptin (protease inhibitor), 1% Triton x-100 (destruction of cells), 1% Sodium deoxycholate (moderate denaturing agent and protein solubilizer) ), 0.1% SDS (strong denaturing agent and protein solubilizer). 7M urea, 2M thiourea (can improve the melting of membrane proteins), proteinase K, etc.

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