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Analysis of problems in the use of lysate guanidine isothiocyanate |593-84-0|

2017-03-01 来源:亚科官网
1 March 2017
DNA提取
  Guanidine isothiocyanate is a strong protein denaturant, not only can quickly dissolve protein, resulting in cell structure broken, nuclear protein due to the destruction of its secondary structure and quickly seperates with the nucleic acid, but also has a strong denaturation of RNase. Thus, guanidine isothiocyanate can be used to denature cells, isolate RNA from proteins, and release RNA into solution. However, in the preparation and use of guanidine isothiocyanate solution, there often are some problems, this paper will summarize these issues.
Should the solution of guanidine isothiocyanate be preparation before use?
  Make the final concentration of guanidine isothiocyanate of 4M as an example: 42mM sodium citrate, 0.83% N-lauryl sarcosine (sodium dodecyl, N-methylglycine), 0.2mM β-mercaptoethanol to make the CSB buffer solution; guanyl isothiocyanate 25g, CSB buffer 33mL, mixed until completely dissolved, it can be heated to 65℃ to help dissolve, to obtain the denaturation solution, and it should be preserved at 4℃.
  Therefore, the guanidine isothiocyanate solution may not have to prepare before use, and there are solutions with different concentrations selling on the market, but the storage period should not be too long, otherwise there will be precipitation, and the experimenter need to carefully check before use; it is recommended to subpackage if the solution is too much.
Is it necessary to sterilize the guanidine isothiocyanate solution after preparation?
  The guanidine isothiocyanate solution does not need to be sterilized. The solution is not very stable, and the lysate is usually treated with 0.1% DEPC water and the water has been sterilized. In addition, all reagents that extract RNA need to be formulated with DEPC water, and the reagents are preferably fresh and only open for RNA.
  Although the lysate solution does not require sterilization, but it should be noted that the plastic products used in the experiment, which marked with Rnase-Free, if not used before, can be used directly, the others must be sterilized: adding DEPC to double distilled water, make the final concentration of 0.1%, immerse the plastic products into the solution, stay overnight in ventilation Fume, and then sealed with aluminum foil, High temperature and high pressure sterilization for at least 30min.
What is the toxicity of guanidine isothiocyanate?
  Inhalation, ingestion, and skin contact with guanidine isothiocyanate can cause damage. Therefore, it is necessary to wear experimental clothing, gloves and goggles during operation. Its contact with acid can release extremely high toxic gas, which is harmful to aquatic life. It has a long-term adverse effect on the water environment and should not be released to the environment.
How to deal with the waste?
  Laboratory pollution is mainly biological pollution and chemical pollution, according to the shape can be divided into waste water, waste gas and solid pollutants. Among them, biological pollution includes biological waste pollution and biological bacterial toxins pollution, and chemical pollution includes organic pollution and inorganic pollution. In general, all kinds of waste should be classified, in principle, it should be recycled to the original bottle. If mixed, experimenter need to make sure the reagents does not produce heat after mixing, toxic gases, explosions and so on. The name and concentration of the solutions should also be indicated on the recycled bottles, in order ro avoid environmental pollution and poison.
  With the rapid development of molecular biology, genetic diagnosis has become the most accurate and effective means of detection, the process of gene diagnosis, the virus nucleic acid extraction is a prerequisite for detection, nucleic acid extraction quality also affects the accuracy of detection, and the quality of the lysate directly affects the quality of nucleic acid extraction, which in turn affects the experimental results. The guanidine isothiocyanate solution is the most commonly used cell lysate, and only the details of each step are handled, the best experimental results can be obtained on the basis of safety.
Edited by the Editorial Office of Suzhou Yacoo Science Co., Ltd.