Precautions for RNA extraction liquid nitrogen polishing tissue

Liquid nitrogen grinding step


 

1, take the mortar out of the oven, use a special container to hold some liquid nitrogen for use;

2, carefully add liquid nitrogen and soak the mortar, soak the pre-cooled mortar and mortar (when adding liquid nitrogen, slowly add to prevent liquid nitrogen from splashing);

3. When the first addition of liquid nitrogen has completely evaporated, slowly add about 1/2 of the mortar volume of liquid nitrogen to the mortar (if the tissue block is large or more, you can soak the mortar), Quickly transfer tissue to liquid nitrogen to ensure that the tissue is completely immersed in liquid nitrogen;

4. Use a pestle to gently tap the tissue in liquid nitrogen to break the tissue into small pieces. Be careful not to use excessive force to prevent the tissue pieces from splashing out of the mortar. When the liquid nitrogen is about to evaporate, use a pestle to Tissue samples are crushed and ground (note that if it is a plant sample, the tissue can be crushed directly with a pestle);

5. Immediately after the liquid nitrogen has evaporated, add 1/3 of the mortar nitrogen (note that the rate of adding liquid nitrogen should be slow, do not make the impact of liquid nitrogen too large, and flush the tissue sample out of the mortar), quickly Grind the tissue with a pestle. According to the degree of change of the tissue state, in order to ensure that the tissue is always in a frozen state, timely replenish with liquid nitrogen for grinding (usually 30s ~ 1min, once at least, more than 3 times), until the tissue is ground into Powdery.

6. Immediately after the tissue is ground into a powder, add an appropriate volume of lysate to lyse it. If the lysate is frozen on the inner wall of the mortar, tap it gently with a pestle, continue to grind the tissue powder and lysate, and grind Sampling of the next sample after the paste is formed;

7. Allow to stand until the sample and lysate mixture melts, transfer the sample that has been melted to a liquid state into a centrifuge tube, and insert it on ice for temporary storage.


 

Other considerations

1. Just add 1-2 times of liquid nitrogen during pre-cooling. Too many times of adding liquid nitrogen will cause frosting of the mortar / pestle and dilute the lysate. (Similarly, during the grinding process, fast grinding is required to reduce the amount of liquid nitrogen added. Number of times, for tissues that are difficult to grind, if frost on the mortar and pestle, wipe the frost with a clean dust-free paper towel, taking care not to touch the tissue);

2. The sampling process should be fast and accurate. Do not expose the sample to the air for a long time. The tweezers that contact the sample need to be sterilized in advance. Wipe with 75% alcohol before use to avoid cross-contamination.

3. During the grinding process, liquid nitrogen must be added in time. The amount of liquid nitrogen can be immersed in the tissue surface to ensure the low temperature of the tissue;

4, the grinding process should be ground in one direction, the tissue should be ground into powder as much as possible plus lysate;


 

5. Liquid nitrogen and lysing solution are harmful to the human body. Pay attention to avoid direct skin contact. It is forbidden to wear shorts, sandals and other clothing that will expose the skin in this experiment. If necessary, you can wear goggles to protect the face.

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