Formaldehyde denaturing gel electrophoresis to identify RNA experiment

Summary

The purpose of this experiment is to learn how to identify RNA by formaldehyde denaturing gel electrophoresis. Agarose gel electrophoresis is often used for the identification of large molecules, but it contains RNase activity, so formaldehyde denaturing gel electrophoresis is used for the identification of RNA in order to denature RNase without denaturing RNA.

Operation method

gel electrophoresis

Principle

Agarose gel electrophoresis is often used to identify large molecules, but it contains RNase activity, so formaldehyde denaturing gel electrophoresis was used to identify RNA, because it is necessary to denature RNase without denaturing RNA.

Materials and Instruments

RNA
MOPS Sodium acetate EDTA Sucrose EDTA Bromophenol blue Xylene cyan Formaldehyde
Electrophoresis Tank Electrophoresis Instrument

Move

1. Preparation of the gel: agarose concentration of 1 . 2%, add 5 × formaldehyde gel electrophoresis buffer and formaldehyde, so that the final concentration of the two are 1 × formaldehyde gel electrophoresis buffer and 2.2 mol / L, respectively; 2.
2. Take a certain amount of RNA sample, add 5 × formaldehyde gel electrophoresis buffer 2.0 μl, 37% formaldehyde gel electrophoresis buffer 2.2 mol/ L. 2. Take a certain amount of RNA sample, add 5 × formaldehyde gel electrophoresis buffer 2.0 μl, 37% formaldehyde 3.5 μl, deionized methyl chloride 3.5 μl, and deionized methyl chloride 3.0 μl. 5 μl, deionized formamide 10.0 μl. 0 μl, slightly centrifuged and mixed in a 65 ℃ water bath for 15 min, and then quickly placed in an ice bath for 5 min; add 2.0 μl of sample buffer. Add 2.0 μl of sample buffer and centrifuge for a while.
3. Before sampling, the gel was pre-electrophoresed in 1 × formaldehyde gel electrophoresis buffer for 5 min (voltage 5 V/cm).

4 . Add the samples into the sample wells of the gel, and electrophoresis was carried out at a voltage of 4 V/cm; mix the anode and cathode solutions every 30 min, and then continue the electrophoresis. 5.
5. After electrophoresis, put the gel into a 0.5 μg/ml solution. 5. After electrophoresis, put the gel into 0.5 μg/ml EB solution and stain it for 30 min; observe the gel under ultraviolet light.

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