How is the cell lysis buffer prepared?
Scientists use lysis buffer when extracting DNA or protein from cells for analysis, especially in the case of bacteria. Lysis buffer helps to destroy open cells, so its contents can be changed. Some examples include salts, detergents, chelating agents and inhibitors, and some alkaline chemicals. In the following, I will discuss with you the principle of lysis buffer preparation?
1. Tris-HCL buffer can stabilize the pH value during cell division. Tris-HCL is one of the common buffer chemicals at pH 8. HEPES is another common buffer chemical in these experiments. Sodium chloride salt can also increase ionic strength, which is the total concentration of solutes outside the cell.
2. The lysis buffer usually also includes a chelating agent, such as ethylenediaminetetraacetic acid (EDTA) or ethylenediaminetetraacetic acid (EGTA). These chemicals combine with two positively charged metal ions, making them unusable for other reactions. Many DNA enzymes (proteins that chew DNA) and proteases (proteins that break down other proteins) require magnesium ions to function, so EDTA and EGTA deprive them of this key component and help reduce the level of protease or DNAse activity. However, they cannot completely rule out the fact that certain proteases do not rely on magnesium cofactors, so the lysis buffer sometimes contains chemicals called protease inhibitors, which bind to proteases and prevent them from working properly.
3. Alkaline lysis is a commonly used technique to purify plasmids from bacteria, involving three solutions. The first one contains glucose, tris-HCL buffer and EDTA. Glucose produces a high concentration of solutes outside the bacteria, so they become a little relaxed, making it easier to lyse. The functions of EDTA and tris-HCL are as described above. The second solution is actually to lyse the cells. This one contains SDS detergent and NaOH, which can raise the pH to 12 or higher, denature the protein in the cell and separate the DNA into single strands. The third solution contains potassium acetate, which restores the pH to a more neutral level, so the plasmid DNA strands can reassemble. At the same time, the denatured protein will coalesce and precipitate.
In summary, everyone should have some understanding of the application and classification of lysis buffers, and Tris-HCL buffer is the most used component of the lysis buffer, but because this buffer has more applications, As a result, there are not many stocks on the market, which makes buyers very distressed. Here I recommend our company, Hubei Xindesheng Material Co., Ltd., a manufacturer specializing in the production of biological buffers. The company has been established for more than ten years and has a wealth of product technology experience. High production efficiency, excellent product quality, fast delivery speed, after-sales guarantee, customized services and testing services can be provided according to customers, and professional technical support is provided. In any case, trust our company, it must be your purchase of biological buffers. Two choices.
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