First, the purpose of the experiment
Learn how to use RNA agarose gel electrophoresis.
Second, the experimental principle
RNA can be detected using non-denaturing or denaturing gel electrophoresis. In non-denaturing electrophoresis, RNA molecules of different molecular weights in the mixture can be separated, and the molecular weight cannot be determined. Only in the case of degeneration, the RNA molecule is fully stretched and its mobility is proportional to the molecular weight.
Determining the integrity of RNA extracts is one of the main purposes of electrophoresis. The electropherogram of intact undegraded RNA preparations should clearly see the three bands of 18s rRNA, 28s rRNA, and 5s rRNA, and the brightness of 28s rRNA should be twice that of 18s rRNA.
Third, materials, reagents and appliances
1, material
Total RNA extract.
2, reagents
(1) 0.1% DEPC water: 200 ml of double distilled deionized water plus 0.2 ml of DEPC coke acid and diethyl ester were mixed, left at room temperature overnight, and autoclaved.
(2) 10x running buffer.
Morpholinopropane sulfonic acid (MOPS) 0.4 mol/L (pH 7.0)
NaAc 0.1mol/L
Ethylenediaminetetraacetic acid (EDTA) 10mmol/L
(3) 50mL denaturing agarose gel (1%)
(4) 10x running buffer 5 mL
Agarose 0.5 g
0.1% DEPC water 36.5 mL
Heat to dissolve, slightly cool, add 8.5 mL of 37% formaldehyde.
(5) Loading buffer: 50% glycerol, 1 mmmol/LEDTA, 0.4% bromophenol blue, 0.4% xylene blue.
(6) Formamide (deionized).
3, appliances
(1) Electrophoresis system
(2) UV perspective
Fourth, the operation steps
1. Rinse the gelatinous appliance with 70% ethanol and let it dry.
2, making glue:
0.5 g of agarose powder was weighed and added to an Erlenmeyer flask containing 36.5 mL of DEPC water, and heated to completely dissolve the agarose. After a little cooling, 5 mL of 10x running buffer and 8.5 mL of formaldehyde were added. Then, the gel is filled in the glue tank, the comb is inserted, and it is placed horizontally to be solidified and used.
3, loading:
The following reagents were mixed in a small clean centrifuge tube: 2 μl of running buffer (10x), 3.5 mL of formaldehyde, 10 mL of formamide, and 3.5 μl of RNA sample. Mix well, keep at 60 ° C for 10 min, and cool on ice. Add 3 μl of the loading buffer and mix well, and apply an appropriate amount to the gel sample well. At the same time, point the RNA standard sample.
4, electrophoresis:
Open the electrophoresis apparatus and adjust the voltage to 7.5V/cm.
5. After the end of electrophoresis, observe by ultraviolet fluoroscopy.
Five, matters needing attention
RNase contamination must be maintained in this experiment to avoid RNA degradation. All reagents were prepared in DEPC water and the equipment was also rinsed with DEPC water and sterilized.
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