Experimental principle After extracting the total RNA of the sample, the quality of the RNA is generally judged based on the gel electrophoresis map of the RNA. Because RNA easily forms secondary structures, formaldehyde denaturing gel is often used for RNA electrophoresis, and the resulting electrophoresis can truly reflect the quality of RNA. Separate the RNA in the gel by gel electrophoresis, identify it by adding standard nucleic acid molecules (markers), and use it for membrane transfer and hybridization. Experimental reagent 1. DEPC water treatment: Take 2L of deionized water with a measuring cylinder, add 2ml of DEPC to 2L of deionized water in a fume hood, and the final concentration of DEPC is 0.1%. Close the lid quickly, mix well, then place in a shaker at medium speed for at least 4 hr, and then autoclave. During sterilization, loosen the bottle cap and sterilize 15 pounds for 20 minutes. 2. 10 × MOPS buffer (ie 10 × FA buffer): 500ml 0.2mol / L MOPS (3- [N-morpholine] propanesulfonic acid) 80mmol / L NaAc 10mmol / L EDTANa2 After dissolving 20.6g MOPS and 4.1g NaAc in 400ml DEPC water, add 10ml 0.5mol / L EDTA to make a fixed volume of 500ml. Filter and sterilize with a 0.22mm filter, put it in a brown bottle and store at room temperature in the dark. Or 0.2mol / LMOPS (3- [N-morpholine] propanesulfonic acid) 50mmol / L NaAc 10mmol / L EDTA Adjust the pH to 7.0 with 2mol / L sodium hydroxide and dilute with DEPC water before use. 3. 1 × MOPS electrophoresis buffer (ie 1 × formaldehyde denaturing gel electrophoresis buffer 1 × running buffer): 1500ml 10 × MOPS 150ml 37% formaldehyde 80ml Add water to 1500ml. Generally, the electrophoresis buffer needs to be changed after 3 times of electrophoresis. 4. 5 × formaldehyde denaturing gel loading buffer (5 × loading buffer): 10ml Prepare water-saturated bromophenol blue solution in advance: add about 0.1mg bromophenol blue to a 1.5ml centrifuge tube, add 1ml of DEPC water to dissolve, shake thoroughly to dissolve, centrifuge, it can be seen that there is bromophenol blue powder at the bottom of the centrifuge tube Bromophenol blue liquid saturated with water. In a 15ml sterilized centrifuge tube, add the following ingredients in sequence: 4.0ml 10 × FA buffer 3.1ml formamide 2.0ml 100% glycerin 720ul 37% formaldehyde 80ul 0.5MEDTA (PH 8.0) 16ul water saturated bromophenol blue 100ul DEPC water Mix well, aliquot and store at -20 ℃, usually at 4 ℃ Or formaldehyde gel denaturing RNA electrophoresis loading buffer (10 ×): 10 50% glycerin 1mmol / L EDTA 0.25% bromophenol blue 0.25% xylene cyanide 5ml glycerin, 20ul 0.5mol / L EDTA, 25mg bromophenol blue, 25mg xylene cyanide, add DEPC water to 10ml, store at 4 ° C after high pressure. Laboratory equipment 1. Electrophoresis equipment 2. Ultraviolet lamp Experimental procedure 1. The electrophoresis tank was soaked with 0.3% H2O2 for 30 minutes, rinsed with DEPC water to dry. 2. Spreading glue (40ml) Agarose 0.55g DEPC water 36ml 10 × MOPS 5ml 37% formaldehyde 9ml Weigh 0.55 grams of agarose plus 36 ml of DEPC water, heat the microwave to dissolve the agarose, and visually observe no particulate suspension. Cool to 50-60 ° C, add 9ml of formaldehyde (37%) and 5ml of 10x electrophoresis buffer, then pour the gel, insert a comb of appropriate length and width, and leave it at room temperature for more than 30 minutes to solidify the gel. 3. For RNA sample processing (take one lane as an example), generally take 0.3 g of total RNA, add 1/5 volume of 5 × loading buffer, heat at 65 ° C for 5 min, and quench on ice to eliminate the secondary structure of RNA. It is recommended to add 0.5-1.0 ul of ethidium bromide (EB, concentration 1.0 mg / mL) to the RNA sample before loading, rather than adding EB to the gel, so that the background after electrophoresis is low. The prepared formaldehyde-modified gel is pre-electrophoresed in 1 × formaldehyde-modified gel electrophoresis buffer for 15 minutes. RNA samples were electrophoresed at a voltage drop of 5-10 V / cm for 30 min. 4. Add 2.0ml formaldehyde gel loading buffer (10 ×) 5. Loading and electrophoresis The gel was pre-electrophoresed for 15 min, and the voltage was reduced to 5-10 v / cm. Then add samples and standards, electrophoresis at a voltage drop of 3-4v / cm, the electrophoresis solution is 1 × MOPS electrophoresis buffer. Until bromophenol blue migrated to the 3/4 downstream of the glue. 6. After the electrophoresis, under the UV lamp, carefully measure the distance between 28S rRNA and 18S rRNA to the sample well, and take a picture together with the fluorescence ruler to record the position of the standard Precautions 1. Each lane can analyze up to 30mg RNA, usually 10-20mg total RNA for Northern hybridization, which can detect high abundance mRNA (accounting for more than 0.1% of total mRNA); if the amount of RNA to be tested is extremely small, add 0.5 -3.0mg poly (A) RNA. 2. Standards 28S rRNA ≥6333 base; 18S rRNA ≥2366 base; 9S rabbit b globin mRNA ≥710 base. Bromophenol blue is in the front (≥300bp), xylene cyanide FF is in the back (≥4kb). Fixed furniture,loose furniture,for decoration,furniture villa Work with Leading Villa PinSheng hotel furniture , https://www.pinshotelfurniture.com