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4.) Add ethidium bromide (IB40075) to the diluted electrophoresis buffer to a final concentration
of 0.5μg/ml.
NOTE: The addition of ethidium bromide to both the gel and the running buffer will result in
maximum detection levels by providing high levels of sample fluorescence with an evenly
low level of background.
5.) Add 6.6ml of the 1X electrophoresis buffer containing ethidium bromide made in step 4 per
millimeter of gel thickness desired, up to a maximum of 50ml, to the flask containing the
agarose (IB70035-40-42-45). A 50ml gel solution will make a 7.6mm thick gel. Thinner gels
may be made, however care must be taken that the wells are deep enough to accommodate the
desired sample volume.
Catalog #
Comb Description
IB51040
1.0mm, 8 tooth
IB51045
1.0mm, 12 tooth
IB51050
1.0mm, 15 tooth
IB51055
1.5mm, 8 tooth
IB51060
1.5mm, 12 tooth
IB51065
1.5mm, 15 tooth
IB51070
1.0mm, 0 Marker, 1 Sample
IB51075
1.0mm, 1 Marker, 1 Sample
IB51080
1.5mm, 0 Marker, 1 Sample
IB51085
1.5mm, 1 Marker, 1 Sample
6.) Make note of the total solution volume so that a degree of evaporation can be determined and
corrected for.
7.) Heat the agarose slurry in a microwave oven for 90 seconds. Swirl the flask to make sure any
grains sticking to the walls enter into the solution, undissolved agarose appears as small
"lenses" floating in the solution. Heat the solution for an additional 30-60 seconds.
Re-examine the solution and repeat the heating process until the agarose completely dissolves.
8.) Add deionized water to replace any volume lost through evaporation during the heating process.
P
REPARATION OF THE
RNA molecules are separated by electrophoresis through denaturing gels prior to analysis by northern
hybridization. Agarose gels containing formaldehyde are commonly used for RNA electrophoresis.
Presented below is a general protocol for electrophoresis of RNA using formaldehyde gels.
CAUTION! All equipment and solutions used in the following protocol should be treated with DEPC
(diethyl pyrocarbonate) or acetic anhydride prior to use to inhibit RNase activity. It is
recommended that dedicated solutions be made solely for RNA work to minimize the risk
of sample degradation due to RNase activity.
NOTE: Staining RNA samples with ethidium bromide has been reported to reduce sample blotting
efficiency. Therefore, if samples are to be analyzed by northern hybridization after elec-
trophoresis, run a duplicate lane(s) for staining, or minimize the exposure of RNA samples
to ethidium bromide by following the post-electrophoresis staining protocol on page 10.
Proceed to "Casting the Gel" on page 6.
A
G
- RNA
GAROSE
EL
Sample Volume
Well Width
4.7mm
2.8mm
2.2mm
4.7mm
2.8mm
2.2mm
62.6mm
4.7mm, 54.7mm
62.6mm
4.7mm, 54.7mm
Per mm Gel
4.7ul
2.7ul
2.3ul
7.0ul
4.0ul
3.3ul
62.6ul
4.7ul, 54.7ul
94ul
7.0ul, 82ul
5
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