Experimental procedure
1. All containers are autoclaved twice, DEPC is autoclaved, all reagents are prepared with DEPC and sterilized at high temperature.
2. In a sterile 2 mL tube, add: 5 mol/L guanidinium isothiocyanate 0.7 mL, phenol 0.4 mL, and NaAc (pH 4.0) 0.1 mL.
3. Weigh the leaves of 0.2g wheat yellowing seedlings, quickly grind them into powder in liquid nitrogen, transfer them to the prepared centrifuge tube, and violently oscillate.
4. Mix and ice for 30 minutes.
5. 4 ° C, 12000 rpm, 10 min.
6. Add 0.4 mL of chloroform to the supernatant tube and shake vigorously. Place in an ice bath for 5 min.
7. 4 ° C, 12000 rpm, 10 min.
8. Discard the organic phase (lower layer) Add 0.4 mL of chloroform and 0.4 mL of phenol and let stand for 5 min at room temperature.
9. 4 ° C, 12000 rpm, 10 min, discard the organic phase and add an equal volume of isopropanol. Leave at -20 ° C for 1 h.
10. 4 ° C, 12000 rpm, 10 min, the precipitate was washed twice with 70% ethanol.
11. The room temperature is slightly dry.
12. Precipitate and dissolve with 20μLDEPC water (preserved at -20 °C)
13. RNA electrophoresis: 1% agarose gel 20mL, 8μL sample, 1μL sample buffer, 1μLSYBR, 100V constant pressure electrophoresis, OD260 and OD260/OD280 were measured.
Precautions
1. The purpose of step 1 is to remove the effects of RNase (exogenous). Sterilization twice can destroy the renaturation process of RNase. Although RNase is highly reproducible, two consecutive high temperatures can disrupt its renaturation process, thereby inactivating RNase. DEPC is a non-competitive inhibitor of RNase. DEPC can bind to N on the histidine ring of the active center of RNase, thereby inactivating RNase. Since DEPC binds to the N of the RNA to modify the RNA, the DEPC must be finally removed. At high temperature sterilization, DEPC volatilizes due to pyrolysis. UV can also modify RNA and should be avoided during the experiment. DEPC is called DEPC water after sterilization and it is DEPC free. During the experiment, the gloves should be changed frequently, and if necessary, in the ultra-clean workbench.
2. The denaturant must be prepared in advance. Isothiocyanate can denature RNase or break cells. This test does not allow cell disruption by enzymatic methods. Because of the suitable conditions for proteinase K, RNase can also be made active. The acidity of NaAc allows the DNA to be in the organic phase (phenolic phase), while under alkaline conditions, both DNA and RNA are in the aqueous phase and cannot be separated. Phenol is used to extract DNA and proteins.
3. Low temperature prevents endogenous RNase from degrading RNA. The violent shock is to spread all the cells and immerse them in the denaturant. Low temperature cells form a mass in a relatively high temperature liquid, which causes the internal RNA to have no chance of hydrolyzing RNA. Because of the lack of photosynthesis, yellowing seedlings of wheat contain less sugar and are easier to extract.
4. The purpose of step 6 is to remove the lipids.
5. The purpose of step 8 is to remove lipids and proteins.
6. The purpose of step 9 is to remove the sugar and dehydrate it. Because of the high salt (2 mol/L NaAc), the RNA can be precipitated.
7. Step 10 Note that because the amount proposed is small and may not be visible, the centrifugation should have a directional mark.
8. RNA is very poorly soluble, so it can only be slightly dry. If it does not dissolve it is because there are too many sugar residues.
9. DEPC water should be free of RNase.
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