Liquid Nitrogen Grinding

Summary

Liquid Nitrogen Milling for Applications Related to Deoxyribonucleic Acid (DNA) Isolation

Principle

Molecular biology is the current frontier of life science field, its theory and technology has penetrated into the field of various disciplines, and molecular biology experiments in life science research is quite important. In practice, many experimental materials, such as muscle tissue, connective tissue, bone tissue, hair tissue, etc. on animals, plant roots, stems, leaves, seeds, etc., need to be ground, and then its composition, genes and proteins, and the structure and function of the study.

Liquid nitrogen grinding is widely used in molecular biology experiments, especially for the separation of ribonucleic acid (RNA), deoxyribonucleic acid (DNA). Liquid nitrogen temperature of a 196 ℃, it can make a variety of tissue components are not easy to be destroyed or degradation, but also can make the organization hard, brittle increase easy to grind. It is because the temperature of liquid nitrogen is extremely low, the operation should be particularly careful. Liquid nitrogen grinding, one is to terminate all biological reactions inside and outside the cell, such as the extraction of RNA, to prevent the degradation of RNA enzyme reaction, etc.; There is also a reason, that is, the cells in liquid nitrogen is completely frozen hard, grinding can be achieved to achieve a very good effect of breaking the cells, the cells will be ground into a powder, so that the inside of the material released.

Appliance

Isolation of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA)

Operation method

Liquid Nitrogen Grinding

Materials and Instruments

【Materials】Tissue
[Apparatus, Consumables] Mortar and pestle, forceps, scissors, 75% alcohol, dust-free paper towel, special container for liquid nitrogen.

Move

Preparation of liquid nitrogen containersLiquid nitrogen is usually stored in a liquid nitrogen tank. Before the experiment, prepare a thermos bucket, transfer the appropriate amount of liquid nitrogen to the thermos bucket, and also prepare a stainless steel or plastic spoon, if the handle of the spoon is not long enough, find a way to lengthen it, so as to avoid frostbite hands.Tissue handling and dosageIf any tissue is not used immediately after obtaining it, it should be stored in a low-temperature environment as soon as possible (quick-freezing in liquid nitrogen at 196℃, preservation at 70℃). When used, plant tissues should be first washed with tap water to clean the appearance of sediment, rinsed twice with single-steam water, soaked in 75% ethanol for about 30 seconds, then rinsed twice with sterilized double-steam water, and then blotted with sterile filter paper, and then take the appropriate part of the tissue according to the need. Animal tissues need not be washed, but the surrounding tissues and blood clots should be removed as much as possible. The amount of tissue should be 1-2 times larger than the actual amount needed, because some of the tissue will stick to the mortar and rod when grinding, which will result in the loss of some tissue samples.Fixation of the mortarWe found that the temperature of the mortar is very low after adding liquid nitrogen, and many ice crystals will be formed on the surface, so it is easy to slip when grinding the tissues with force, and the liquid nitrogen mixture inside the mortar will be easily spilled, resulting in the loss of tissues and easy to frostbite hands. If some gauze or filter paper under the mortar, although not easy to slide, but because of the cushioning effect, grinding is not force, not desirable. Later, we made the following improvements: one is to do a wooden support, that is, choose a piece of wood about two inches thick, dug a hemispherical hole in the top (size according to the size of the mantle depending on the size of the), the ends of the wooden board to leave a certain length to prepare for the hand to hold the use of the mantle will be used to research the mantle stuck in a hemispherical hole, not only stable but also will not be frostbitten hand; two is to prepare a clean towel or gauze (dry), folded into a long strip, the mantle is surrounded by the ends of the merger! After grabbing the hand, this method is simple and practical, but be especially careful to avoid frostbite.Grinding methodEmpty bowl first pre-cooled with liquid nitrogen, and then the upper end of the rod wrapped in gauze (force when the hand will not hurt), it is best to cut the tissue into thin slices, grinding is not easy to spatter, but also easier to grind, the rod is best against the palm of the hand, so as to facilitate the force. Avoid drying out the liquid nitrogen during the grinding process, and grind until it is a fine powder. When the liquid nitrogen has just evaporated, use a small medicine spoon to take out the required amount for the next experiment, or put the tissue powder into a container and store it at a temperature of 70℃ for the next experiment. Proper use of the liquid nitrogen grinding method will lay the foundation for the success of the next step of the experiment. It is worth mentioning that all contacting vessels and consumables should be cleaned and sterilized during the operation. If the milled tissues are used for RNA extraction, the vessels and consumables must be inactivated by diethylpyrocarbonate (DEPC) and autoclaved.

Caveat

Put the sample into the mortar, pour a small amount of liquid nitrogen, the liquid nitrogen will be like boiling, this time quickly grinding. The action must not be too large, it is best to use the force of the palm of the hand downward rotating grinding, too much action will splash the sample, if the sample is very small amount of direct may be ground out. About a few seconds of boiling is over, a few more grinding can be added to the liquid nitrogen, repeat the above process, until you think the grinding is relatively fine.The temperature of liquid nitrogen at atmospheric pressure is quite low, once it comes into contact with the surface of an object, it will boil rapidly, and at the same time, it will take away a large amount of heat energy. Therefore, extra care should be taken when using liquid nitrogen to avoid direct contact with skin.Protective gear should be worn when loading, e.g. anti-freeze gloves. Do not use cotton gloves, cotton gloves will absorb a large amount of liquid nitrogen by capillary phenomenon and increase the possibility of frostbite (even more dangerous than not wearing).The mortar should be made of porcelain, of course, the same mortar and pestle, the amount of liquid nitrogen depends on the size of your mortar and the amount of experimental materials, it is recommended that you use a smaller mortar, so that when grinding the material is more concentrated and good to study a little bit of liquid nitrogen can be poured into the mortar volume of half of the mortar.

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