Innovation of efficient cell breaking method for yeast
——The ultimate grinding method: freeze grinding method
Yeast is a universal host for gene expression studies and recombinant protein production. However, traditional enzymatic hydrolysis of yeast mRNA and content proteins is often very difficult. We used cell total RNA extraction as an example to compare various yeast cell wall breaking methods. We used different methods to break the yeast, then extracted the RNA with Trizol reagent, and finally carried out electrophoresis detection. It was found that the effects of different methods of breaking the wall were as follows:
SPEX's Freezer/Mill freeze grinder further improved the above-mentioned liquid nitrogen grinding method, making the experiment of breaking the yeast more simple and unattended. The sample is placed in a closed grinding container, immersed in liquid nitrogen, electromagnetic oscillating technology is used, electromagnetically reciprocally drives the steel hitter, oscillates the installed sample, the kinetic energy inertia is larger, and the high kinetic energy impactor is hit by a large area, and the pulverization is rapidly pulverized. During the process, the sample is always at liquid nitrogen temperature (-196 ° C).
experimental method:
Step 1: Pre-freeze the sample grinding jar.
The second step: adding the yeast liquid to the liquid nitrogen to control the speed, so that the bacterial liquid enters the liquid nitrogen to form small particles. The bacterial liquid nitrogen particles are transferred to a grinding tank. Place in the sample compartment of the freezer.
The third step: set the grinding program, pre-cooling for 10-15min, grinding for 2min, 15 cycles, and intermittent between the two grindings for 1min.
Experimental results:
It has been tested that more than 90% of yeast cells can be broken by this method. Yeast RNA and intracellular proteins remain highly active.
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