Formaldehyde Agarose Gel Electrophoresis

 

 

The following protocol for formaldehyde-agarose gel electrophoresis gives enhanced sensitivity for gel and subsequent analysis (e.g. northern blotting). A key feature is the concentrated RNA loading buffer that allows a larger volume of RNA sample to be loaded onto the gel than conventional protocols (e.g. Sambrook, J. et al., eds. (1989) Molecular cloning a laboratory manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press).

 

1.2% Formaldehyde Agarose gel preparation

To prepare Formaldehyde Agarose gel (1.2% agarose) of size 10 x 14 x 0.7 cm, mix:

1.2 g agarose

10 ml 10x Formaldehyde Agarose gel buffer (see composition below)

Add RNase-free water to 100 ml

If smaller or larger gels are needed, adjust the quantities of components proportionately. Heat the mixture to melt agarose. Cool to 65C in a water bath. Add 1.8 ml of 37% (12.3 M) formaldehyde (toxic) and 1 l of a 10 mg/ml ethidium bromide (mutagenic) stock solution. Mix thoroughly and pour onto gel support. Prior to running the gel, equilibrate in 1x Formaldehyde Agarose gel running buffer for at least 30 min.

 

RNA sample preparation for Formaldehyde Agarose gel electrophoresis

Add 1 volume of 5x loading buffer per 4 volumes of RNA sample (for example 10 l of loading buffer and 40 l of RNA) and mix.

Incubate for 35 min at 65C, chill on ice, and load onto the equilibrated Formaldehyde Agarose gel.

 

Gel running conditions

Run gel at 57 V/cm in 1x Formaldehyde Agarose gel running buffer.

 

 

Composition of Formaldehyde Agarose gel buffers


10x Formaldehyde Agarose Gel buffer

200 mM 3-[N-morpholino]propanesulfonic acid (MOPS) (free acid)

50 mM sodium acetate

10 mM EDTA

pH to 7.0 with NaOH

 

1x Formaldehyde Agarose Gel Running Buffer

100 ml 10x Formaldehyde Agarose gel buffer

20 ml 37% (12.3 M) formaldehyde

880 ml RNase-free water

 

5x RNA Loading Buffer

16 l saturated aqueous bromophenol blue solution

80 l 500 mM EDTA, pH 8.0

720 l 37% (12.3 M) formaldehyde

2 ml 100% glycerol

3084 l formamide

4 ml 10 x Formaldehyde Agarose gel buffer

RNase-free water to 10 ml

Stability: Approximately 3 months at 4C