4 Electrophoresis of DNA and RNA Fragments
79
To the RNA samples (total RNA 10-20 11g; poly(At RNA 1-2 11g per lane)
add the following solutions:
• 10 111 formamide (deionized)
• 3.5 111 formaldehyde (37%)
• 2 111 10xMOPS buffer *
• 4.5 111 HzO
Denature samples by heating at 65°C for 15 min, then cool them on ice.
Parallel to RNA samples, run 5 11g RNA size markers treated in a similar
manner (Gibco/BRL). RNA can be directly visualized in the gel by adding
1 111 of a 10 mg/ml ethidium bromide solution to the sample mix and denaturing in the presence of ethidium bromide. (Note: ethidium bromide
solution must be prepared using DEPC-treated water.) Staining after the
run is not as efficient as formaldehyde interferes with staining. Alternatively, RNA can be visualized after the transfer to the membrane by UV
shadowing with an intense UV source.
* 10xMOPS
• 0.2 M 3-morpholinopropanesulfonic acid, pH 7.0
• 50 mM sodium acetate
• 10 mM EDTA
Submerge gel in the running buffer (1xMOPS buffer with 3.7% formaldehyde) and load samples. To monitor progress of electrophoresis, add 5 111
dye mix (50% glycerol; 1 mM EDTA; 0.4% bromphenol blue and 0.4% xylene
cyanol) to an unfilled well. Constant recirculation of the buffer is not necessary, but after 1 - 2 h the buffer from each reservior should be mixed.
Remove the gel from the running buffer and wash two times with H20. If the
blot will be probed for transcripts larger than 2 kb long, or if the gel is more
than 1 o/o agarose, perform the following steps:
1. Soak the gel in 50 mM NaOH; 0.15 M NaCl for 20 min.
2. Neutralize the gel in 0.1 M Tris (pH 7.5); 0.15 M NaCl for 30 min.
Denaturation of
RNA sample prior
to loading
Electrophoresis
Transfer of
denatured RNA
to the membranes
79
To the RNA samples (total RNA 10-20 11g; poly(At RNA 1-2 11g per lane)
add the following solutions:
• 10 111 formamide (deionized)
• 3.5 111 formaldehyde (37%)
• 2 111 10xMOPS buffer *
• 4.5 111 HzO
Denature samples by heating at 65°C for 15 min, then cool them on ice.
Parallel to RNA samples, run 5 11g RNA size markers treated in a similar
manner (Gibco/BRL). RNA can be directly visualized in the gel by adding
1 111 of a 10 mg/ml ethidium bromide solution to the sample mix and denaturing in the presence of ethidium bromide. (Note: ethidium bromide
solution must be prepared using DEPC-treated water.) Staining after the
run is not as efficient as formaldehyde interferes with staining. Alternatively, RNA can be visualized after the transfer to the membrane by UV
shadowing with an intense UV source.
* 10xMOPS
• 0.2 M 3-morpholinopropanesulfonic acid, pH 7.0
• 50 mM sodium acetate
• 10 mM EDTA
Submerge gel in the running buffer (1xMOPS buffer with 3.7% formaldehyde) and load samples. To monitor progress of electrophoresis, add 5 111
dye mix (50% glycerol; 1 mM EDTA; 0.4% bromphenol blue and 0.4% xylene
cyanol) to an unfilled well. Constant recirculation of the buffer is not necessary, but after 1 - 2 h the buffer from each reservior should be mixed.
Remove the gel from the running buffer and wash two times with H20. If the
blot will be probed for transcripts larger than 2 kb long, or if the gel is more
than 1 o/o agarose, perform the following steps:
1. Soak the gel in 50 mM NaOH; 0.15 M NaCl for 20 min.
2. Neutralize the gel in 0.1 M Tris (pH 7.5); 0.15 M NaCl for 30 min.
Denaturation of
RNA sample prior
to loading
Electrophoresis
Transfer of
denatured RNA
to the membranes