Methylmercury hydroxide agarose gel electrophoresis
Methylmercury hydroxide reacts with the imine bonds on uracil and guanine in RNA, which are involved in Watson-Crick base pairing. Therefore, methylmercury hydroxide is a potent denaturant that disrupts the secondary structure of RNA. In the presence of methylmercury hydroxide, the electrophoretic mobility of RNA is an inverse function of the logarithm of 1g of its molecular mass. Because methylmercury hydroxide reacts with the free radicals required in the polymerization of acrylamide, it cannot be used in polyacrylamide gel electrophoresis, but only in agarose gel electrophoresis and sucrose density gradient centrifugation.
Operation method
Methylmercury hydroxide agarose gel electrophoresis
Materials and Instruments
4X Methylmercury gel electrophoresis buffer 2X Gel spiking buffer Ethidium bromide Ammonium acetate solution Move (i) Materials and equipment Caveat 1) Methylmercury hydroxide is highly toxic and volatile. It is best handled in a fume hood.2) Methylmercury hydroxide should be added to the gel and not to the methylmercury gel electrophoresis buffer. This compound is uncharged and therefore does not migrate out of the gel quickly. However, if the gel is immersed in the electrophoresis solution during electrophoresis, the methylmercury ions will diffuse out of the gel. To avoid this problem, the level of the electrophoresis buffer can be adjusted so that it stays fully connected to all sides of the gel without spilling onto the gel surface. After sampling is complete and the RNA is in the gel, cover the gel with plastic wrap to prevent it from drying out during electrophoresis. For more product details, please visit Aladdin Scientific website.
Horizontal electrophoresis unit Water bath
1) Horizontal electrophoresis unit.
2) Water bath
3) 4X Methylmercury gel electrophoresis buffer: 200 mmol/L boric acid, 20 mmool/LN2B4O7.H2O,40 mmol/LNa2S04(pH8.0)
4) 2X gel spiking buffer:
lmol/L methylmercury hydroxide 25u1
4X Methylmercury gel electrophoresis buffer 500ul
Glycerol 200ul
Bromophenol Blue 2ug
5)0.5ug/ml ethidium bromide
6)0.lmol/L ammonium acetate solution
(ii) Method of operation
1) Preparation of gel: 1% agarose for RNA greater than or equal to 1kb, 1.4% agarose for RNA less than 1kb. Dissolve agarose in 1X methylmercury gel tunnelling buffer, cool to 55°C, add hydroxide of methylmercury to a final concentration of 5 mmol/L, and then fill the gel.
2) Electrophoresis: Mix an equal volume of 2X gel spiking buffer with RNA solution, spot sample and electrophoresis.
3) Observation of results: After electrophoresis, soak the gel with 0.lmol/L ammonium acetate solution containing 0.4 g/ml ethidium bromide for 30~45 min, then perform RNA staining and observe the results under the ultraviolet light. The ammonium ion converts methylmercury into a charged non-volatile complex and promotes the binding of ethidium bromide to RNA.