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Saturday, 24 December 2011

Formaldehyde Agarose Gel Electrophoresis Protocol

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 65°C in a water bath. Add 1.8 ml of 37% (12.3 M) formaldehyde (toxic) and 1 µl 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 µl of loading buffer and 40 µl of RNA) and mix.
Incubate for 3–5 min at 65°C, chill on ice, and load onto the equilibrated Formaldehyde Agarose gel.

Gel running conditions
Run gel at 5–7 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 µl saturated aqueous bromophenol blue solution†
80 µl 500 mM EDTA, pH 8.0
720 µl 37% (12.3 M) formaldehyde
2 ml 100% glycerol
3084 µl formamide
4 ml 10 x Formaldehyde Agarose gel buffer
RNase-free water to 10 ml
Stability: Approximately 3 months at 4°C

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