1. After UV disinfection for 30 minutes, turn off the UV lamp, turn on the normal working state of the clean bench, and disinfect the operator's hands with alcohol.
2. Place the required medium, trypsin to ensure that the bottle is clean and put it on the work surface, ignite the alcohol lamp, wipe the medium and the trypsin bottle with an alcohol cotton ball, and then align the bottle mouth with the alcohol lamp. Disinfect 2-3 times, unscrew the bottle cap and disinfect the bottle mouth and cap again separately, and place them on both sides of the alcohol lamp. In particular, place the medium bottle on a tilting stand with the bottle mouth aligned with the alcohol lamp and placed it closest to the alcohol lamp. The bottle cap is placed on the other side of the alcohol lamp.
3. Disinfect the bottle mouth on the alcohol lamp 2-3 times. After unscrewing, disinfect the bottle mouth and cap again 2-3 times. Place the bottle on the right side of the alcohol lamp and then hang the culture solution in the bottle. In the dirty tank, disinfect the 2-3 times in the alcohol lamp and put it vertically. Take a 1ml pipette and quickly move it on the alcohol lamp for 1-2 times, then put the tip of the pipe and take 1.5ml trypsin solution and transfer it to the culture bottle.
4. Incubate the cell layer so that the pancreatic enzyme is immersed in the entire bottle wall. The time is about 40s. Immediately after the light is erected, there is a pinhole-like gap between the cells and the cells, and 1-2 cells fall off. The best time for trypsin digestion. If you see that the cells are detached from the bottle wall and trypsinized excessively; if you can't see pinhole-like gaps and cell detachment, you should continue to digest, gently and gently lay the culture flask so that the trypsin is immersed in the cell layer for 1 s. Quickly erect the observation of the cells against the light, which can be repeated several times until there is a pinhole-like gap and an optimal digestion state of 1-2 cells. Pipette the trypsin.
5. Pipette 1ml of cell cryopreservation solution into the culture flask, and pipette a small amount of medium to gently rinse the wall of the culture bottle 5-8 times, so that the adherent cells completely fall off the medium and gently blow it. 3 times, keeping the cells in a single suspension as much as possible.
6. Transfer 1 ml of the cryopreservation solution containing the cells into the new preservation tube, tighten the cap, and mark the experiment.
7. Disinfect the bottle mouth and cap of the medium for 2-3 times, then tighten, extinguish the alcohol lamp, arrange the test bench, take out the experimental reagents and the dirty tank, etc., and close the ultra-clean workbench.
8. Put the seed-preserving tube in the refrigerator at 4 °C for 30 minutes, take it out and put it in the refrigerator at -20 °C overnight, then put it in the refrigerator at -80 °C for 3-4 days the next day, and finally store it in liquid nitrogen irrigation at -196 °C. Long-term preservation.
Note that this process can also simplify the experience gained from the actual operation process: after the end of step 7, the preservation tube is wrapped with dry absorbent cotton and the tape is sealed and placed in the -80 °C refrigerator for 4-5 days, then it can be stored in liquid nitrogen irrigation. . However, the cell strain can also be stored in the -80 °C refrigerator for 2-3 months.
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