Biomedical Engineering Reference
In-Depth Information
A
0.4 M Tr i s
·
HCl, 20 mM sodium acetate, 1 mM EDTA, pH 7.4
B
6% glyoxal (w/v) in Soln. A
0.8 - 1.5% agarose (w/v) in Soln. A
15
C
C
0.8 - 1.5% agarose (w/v) in Soln. A
14
C
0.8 - 1.5% agarose (w/v) in 6 M urea, 15 mM iodoacetate,
pH 7.4
14
D
50% glycerol (v/v), 0.3% bromophenol blue in ddH
2
O
D
50% formamide (v/v), 6.5% formaldehyde (w/v), 0.5 mM
EDTA, 10-mM sodium phosphate, pH 7.4
E
25% glycerol (v/v), 0.5% SDS (w/v), 0.025% bromophenol blue
(w/v), 25 mM EDTA in ddH
2
O
Variant 1: Glyoxal Denaturation
Agelof2-8mm thicknessismadefromSoln.Cinahorizontal
slab apparatus.
The RNA sample is incubated in Soln. B for 1 h at 50
◦
C.After
cooling, the solution is mixed with 1
/
10 of its volume with Soln. D
andloadedintotheslotsofthegel.
Electrophoresis is run for about 18 h at RT with constant voltage
of 25 - 30 V. Electrode buffer is Soln. A, which circulates between
the electrode chambers during electrophoresis.
After the end of the electrophoresis, the nucleic acids are stained
with aqueous EtBr (0.5
µ
/
g
ml) and visualized on a UV transillumi-
nator.
Variant 2: Formaldehyde Denaturation
Agelof2to8mm thickness is made from Soln. C
in a horizontal
slab apparatus.
RNA samples dissolved in Soln. D
are heated to 65
◦
C for 5 min,
cooled and mixed with 1
/
5 of their volumes of Soln. E
.
Electrophoresis is done in a hood with 35 - 60 V (cv) using
circulating Soln. A
as electrode buffer.
Staining and identification of bands are described in Variant 1.
Variant 3: Urea gel
Agelof2-8mm thickness is made from Soln. C
in a horizontal
slab apparatus. Because urea decomposes slightly during heating,
it should be avoided to melt it several times or to keep it melted for
longer periods.
The RNA samples are dissolved in any buffer, the ionic strength
of which has to be between 0.01 and 0.2 and which may content
detergents.
Run electrophoresis with 5 V
/
cm (cv) for 3 - 6 h using the elec-
trode buffer A
.
Staining and identification of bands are described in Variant 1.
15
The agarose concentration depends on the average size of nucleic acid
fragments: the smaller the fragments, the higher the concentration.
