The chemical nature of RNA is much more active than DNA. The 2' hydroxyl group immediately adjacent to the phosphodiester bond on the RNA molecule can be directly utilized by RNase as an active factor, so that RNases can be activated without metal ions. Because RNase is widely present in the environment, especially RNase A, the structure is extremely stable and cannot be inactivated by autoclaving, which is extremely difficult to eliminate and poses a great threat to the integrity of RNA samples. Since the RNase class A enzyme acts on the histidine residue at the active site, it can be inhibited by the histidine alkylating agent DEPC. In order to avoid RNA degradation, RNA-related experiments are strictly following the following rules of operation:
1. Use RNase-free solution;
2. Wear clean disposable gloves and replace them frequently;
3. Establish an RNA work area, using a dedicated pipette and RNase-free tips;
4. Use sterile disposable plastic utensils and avoid using glassware as much as possible;
5. Metal tools can quickly eliminate pollution by burning for a few seconds;
6. The glassware can be baked at a temperature above 180 ° C for several hours, or after soaking for 1 hour with freshly prepared 0.1% (v / v) DEPC or absolute ethanol, autoclave to remove residual DEPC;
7. The vessel containing polycarbonate or polystyrene material such as electrophoresis tank should be immersed in 3% hydrogen peroxide for 10 minutes, then rinsed with plenty of RNase-free water and used.
8. Plastic products can be soaked in freshly prepared 0.1% (v/v) DEPC water overnight, then autoclaved for 1 hour to hydrolyze residual DEPC;
9. Reagent treatment:
1) Add 1 ml of DEPC per liter of solution, shake at room temperature overnight or shake at 37 ° C for 1 hour, then autoclave for 1 hour to hydrolyze residual DEPC;
2) Since DEPC can react with the primary amino group, the solution containing Tris should not be treated with DEPC. Should use a new open bottle (RNA-specific) Tris powder, dissolved in DEPC treated water or Milli-Q water, apply RNA-specific electrodes to adjust the pH value, then autoclave;
3) For compounds that are unstable to heat (such as DTT, nucleosides, manganese salts, etc.), they should be directly dissolved in DEPC treated water or Milli-Q water, and sterilized by filtration using a 0.2 μm filter;
4) Reagents such as anhydrous ethanol and isopropanol are used for RNA after opening; 75% ethanol should be prepared with anhydrous ethanol and DEPC treated water;
5) An RNase inhibitor is added to the reaction system.
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