1. The purpose of the experiment
Master the principles and methods of plant total RNA non-denaturing gel electrophoresis.
2. Experimental principle
RNA electrophoresis can be performed under both denaturing and non-denaturing conditions. Non-denaturing electrophoresis uses 1.0% -1.4% gel, different RNA bands can also be separated, but its molecular weight cannot be judged. Only under the condition of complete denaturation, the migration rate of RNA has a linear relationship with the logarithm of molecular weight. Therefore, when determining the molecular weight of RNA, a denaturing gel must be used. When you need to quickly check the integrity of the total RNA sample, you can prepare an ordinary 1% agarose gel.
3. Experimental materials, appliances and medicines
Mushroom total RNA solution. Electrophoresis instrument, electrophoresis tank, electronic balance, pipette, pipette tip, microwave oven, ultraviolet transmission detector, etc. Agarose, 1XTAE running buffer, 0.5μg / ml ethidium bromide (EB) 10X loading buffer.
4. Experimental steps
(1) Make agarose gel with 1 × TAE electrophoresis buffer, add 1 × TAE electrophoresis buffer to the liquid surface to cover the gel.
(2) On a clean bench, use a pipette to draw 4 μl of the total RNA sample on the parafilm. Add 5μl of 1 × TAE running buffer and 1μl of 10X loading buffer to the experiment table. After mixing, add the spot well carefully.
(3) Turn on the power switch, adjust the voltage to 100V, so that the RNA is electrophoresed from the negative electrode to the positive electrode. After about 30 minutes, the gel is stained in EB staining solution for 5 minutes, and rinsed with water. Observe the results of RNA electrophoresis on the UV transmission detector.
RNA denaturing agarose gel detection
Reagents:
(1) MOPS buffer (10 *): 0.4mol / L morpholinopropanesulfonic acid (MOPS) (Ph7.0), 0.1mol / L NaAc, 10mol / L EDTA.
(2) Sample loading dye: 50% glycerin, 1 mmol / L EDTA, 0.4% bromophenol blue, 0.4% xylene blue.
(3) Formaldehyde.
(4) Deionized formamide. v Electrophoresis tank cleaning: detergent cleaning (usually soaked overnight)-water rinse-ethanol drying-3% H2O2 filling-left at room temperature for 10 minutes-0.1% DEPC water rinse
operating:
(1) Rinse the gel-making equipment with 70% ethanol and dry it for use
(2) Prepare agarose gel.
â‘ Weigh 0.5g of agarose, put it in a clean 100ml conical flask, add 40ml of distilled water, and heat it in a microwave oven to completely melt the agarose evenly.
②After the gel is cooled to 60-70 ℃, add 9ml of formaldehyde, 5ml of 10X MOPS buffer and 0.5ul of ethidium bromide in sequence, and mix well.
â‘¢ Pouring agarose gel.
(3) Sample preparation:
â‘ Take a 500ul small centrifuge tube treated with DEPC, and add the following reagents in order: 10x MOPS buffer 2ul, formaldehyde 3.5ul, formamide (deionized) 10ul, RNA sample 4.5ul, and mix well.
② Place the centrifuge tube in a 60 ° C water bath for 10 minutes, and then place it on ice for 2 minutes.
â‘¢ Add 3ul of loading dye to the tube and mix well.
(4) Sample loading.
(5) Electrophoresis: Add 1XMOPS buffer to the electrophoresis tank, and run at a voltage of 7.5V / ml.
(6) After the electrophoresis is over, check the results under the UV lamp.
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