Formaldehyde denaturation electrophoresis of RNA
Formaldehyde denaturation electrophoresis of RNA
Formaldehyde denaturation electrophoresis of RNA can be used for: (1) after extracting the total RNA of the sample, the quality of RNA is generally judged according to the gel electrophoresis graph of RNA; (2) since RNA is easy to form secondary structure, formaldehyde denaturation gel is commonly used for RNA electrophoresis, and the obtained electropherogram can truly reflect the quality status of RNA.
Operation method
Formaldehyde denaturation electrophoresis of RNA
Principle
The results of nucleic acid electrophoresis traced with fluorescent dyes such as ethidium bromide can be used to determine the purity of nucleic acids. Since DNA molecules are much larger than RNA molecules, the electrophoretic mobility is low; whereas rRNA is the most abundant in RNA, accounting for 80%~85%, tRNA and intranuclear small molecule RNA account for 15%~20%, and mRNA accounts for 1%~5%. Therefore, the total RNA electrophoresis can present characteristic three bands. In prokaryotic organisms, the 23S and 16S rRNA bands and the bands composed of 5S rRNA, 5S and 5.8S rRNA and tRNA are clearly visible. mRNA is generally invisible due to its small amount and different molecular sizes. Therefore, it is possible to analyze the results of nucleic acid gel electrophoresis using ethidium bromide as a tracer dye.
Materials and Instruments
Plant Total RNA Move I. Materials, reagents and instruments Caveat Up to 30 mg of RNA can be analyzed per lane, and Northern hybridization is usually performed with 10-20 mg of total RNA, which allows detection of high abundance mRNA (more than 0.1% of total mRNA); if the amount of RNA to be tested is minimal, 0.5-3.0 mg of poly(A) RNA is added per lane. Common Problems Up to 30 mg of RNA can be analyzed per lane, and Northern hybridization is usually performed with 10-20 mg of total RNA, which allows detection of high abundance mRNA (more than 0.1% of total mRNA); if the amount of RNA to be tested is minimal, 0.5-3.0 mg of poly(A) RNA is added per lane. For more product details, please visit Aladdin Scientific website.
Sample Transport Buffer TAE Buffer Formaldehyde Gel Electrophoresis Buffer Formaldehyde Formamide
Electrophoresis Unit External Transmission Observer
1. Material: plant total RNA 5-10 ug
2. Reagents
(1) Loading buffer50% glycerol1 mmol/L EDTA (pH 8.0)0.25% bromophenol blue25% xylene cyanide FF
(2) 10xTAE Buffer
(3) 5x Formaldehyde Gel Electrophoresis Buffer:0.1 mol/L MOPs (pH 7.0)40 mmol/L sodium acetate5 mmol/L DETA (pH 8.0)
(4) Formaldehyde, formamide
3. Apparatus: electrophoresis unit, UV transmission viewer
II. Experimental Procedures
1. Preparation of formaldehyde denatured agarose gels.
Accurately weigh 2 g Agarose (Sigama) in a 250 mL conical flask, then add 20 mL of 10xTAE Buffer, 144 mLDEPC-treated double-distilled water, melt the gel in the microwave oven, and wait until cooled to 50-60 ℃ and add EB to the final concentration of ≤ 0.5 ug/mL. 36 mL of formaldehyde was added to the formaldehyde in a fume hood, and left for a period of time to reduce the formaldehyde vapor. .
2. Formaldehyde denaturation electrophoresis of RNA
(1) Sample preparationTotal RNA: 10 ug5× formaldehyde gel electrophoresis buffer: 4 ulFormaldehyde: 3.5 ulFormamide: 10 ulAdd into sterile centrifuge tube and mix, denature in 95℃ water bath for 2 min (or 55℃, 15 min), remove and cool in ice.
(2) Add 2 ul of sterile DEPC-treated spiking carrier solution. (The purpose of using spiking carrier solution is threefold: to increase the density of the sample to ensure that the DNA enters the sample wells uniformly; to make the sample colorful, which is convenient for spiking; and to clearly show the swimming position of the sample on the electrophoresis gel. When 0.5 X TBE is used as the electrophoresis solution, bromophenol in agarose swims at the same rate as 300 bp long double-stranded linear DNA, while xylene cyanide FF swims at the same rate as 4 kb long double-stranded linear DNA.)
(3) Submerge the plate in 1x formaldehyde gel electrophoresis buffer and pre-run for 5 min at 5 V/cm before spotting. electrophoresis at 3-4 V/cm after spotting.
(4) At the end of electrophoresis (bromophenol blue migrated to about 8 cm), observe under UV light and take photos.