Agarose-Formaldehyde Electrophoresis

 

Agarose-Formaldehyde Electrophoresis

RNA electrophoresis under denaturing conditions in 2.2M formaldehyde is performed according to Maniatis et al., (1982) using the MOPS buffer system. RNA under these conditions is fully denatured and migrates according to the log₁₀ of its molecular weight.

You will need:-

10 x MOPS buffer :

0.2M MOPS (morpholinopropanesulphonic acid)
50mM sodium acetate
5mM EDTA
The buffer is adjusted to pH 7.0 with 1M NaOH and sterilised by autoclaving.

RNA denaturing buffer :

10ml 100% deionized formamide
3.5ml 40% formaldehyde
1.5ml 10 x MOPS buffer
Formamide is deionized by stirring 100ml with approximately 20g of Amberlite MB3 (or MB1) ion exchange resin for 15 minutes.

N.B: Care should be taken handling solutions containing formaldehyde. Formaldehyde is extremely toxic. Gloves should be worn when preparing and handling solutions containing formaldehyde. Electrophoresis utilising formaldehyde containing buffers sh ould be performed in a well ventilated area i.e. a fume hood.

Agarose is prepared by melting the required amount of agarose in distilled water, cooling to approximately 60°C (hand hot) and adding 40% formaldehyde and 10 x MOPS to give 2.2M formaldehyde and 1 x MOPS, respectively. Example: For 50ml of a 1% agaro se gel, melt 0.5g agarose in 37ml H₂ O, cool to hand hot, add 5ml 10 x MOPS buffer and 8.75ml 40% formaldehyde.

Electrophoresis is in 1 x MOPS, 2.2M formaldehyde.

RNA samples are prepared by adding up to 25mg of RNA in a maximum of 5ul sterile H₂ O, to 15ul RNA denaturation buffer. 1ul 10mg/ml ethidium bromide is added to aid visualisation of RNA after electrophoresis.

N.B.: Care should be taking on removal of the gel forming combs prior to loading of samples as rupture of the well bottoms can occur. Agarose/formaldehyde gels are inherently weaker than the equivalent percentage agarose gels.

Immediately prior to loading, RNA samples are heated to 65°C for approximately 10 minutes to denature any secondary structure, cooled on ice for 2 minutes and 2ul of sterile loading buffer added.

Samples are loaded onto the gel and electrophoresed at no more than 5V/cm, with occasional buffer recirculation, until the leading bromophenol blue dye front has migrated approximately three quarters of the length of the gel. Visualisation of RNA is achie ved by irradiation with short wave (254nm) UV light. Typical markers of RNA quality are 18S (~1900bases) and 28S (~4800bases) RNA molecules.