Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 4)

4. Run the gel under the conditions listed below.
Buffer 1x TBE
Constant power 30 W
Buffer temperature 10 °C
Run time 3.5 hours
Note: Pre-chill the running buffer prior to a run.
5. After the run is completed, the gel can be stained with ethidium
bromide (1 µg/ml) in 1x TBE buffer solution for about 5 minutes or
SyBr Green II (Molecular Probes—1:10,000 dilution) in 1x TBE
buffer solution for about 30–45 minutes. Visualize and photograph
the gel by placing it on a UV transilluminator (Gel Documentation
System 1000, catalog numbers 170-7520 through 170-7527, or Bio-Rad
Polaroid Gel Documentation System, catalog numbers 170-3742
through 170-3749).
6. The result from your gel should look similar to that in Figure 1.
There should be a band shift between the single-strands of the
mutant and wild-type fragments.
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100 bp Molecular Ruler (catalog number 170-8202), on the gel to
approximate the size of the product. The size of the product should
be 299 base pairs in length.
4. Store the amplified DNA at 4 °C. For long term storage (> 2 weeks),
store the amplified DNA at -20 °C.
Section 3
SSCP Gel
In SSCP, a nondenaturing polyacrylamide gel is used to separate
single-stranded DNA. Altered conformation due to a mutation in the
sequence can cause the mutant single-stranded DNA to migrate differ-
ently than the wild-type DNA. This migration difference can be seen as
a band shift between the mutant and wild-type DNA samples.
Note: Refer to the DCode universal mutation detection system manual
for information on correct casting and running an SSCP gel on the
DCode Universal Mutation Detection system.
1. Add 5 µl of the amplified mutant DNA and 5 µl of 2x SSCP gel load-
ing dye to a microfuge tube. Gently mix the contents of the tube. Do
the same for the amplified wild-type DNA.
2. Place the tubes (mutant and wild-type) into a 95 °C water bath for
7 minutes and then on ice for about 5 minutes.
3. Load 10 µl of the samples into the wells of an 8% acrylamide/bis gel
(37.5:1), containing 7% glycerol, and 1x TBE buffer.
Note: Recipe for a 20 x 20 x 0.1 cm gel format.
40% Acrylamide/bis (37.5:1) 8 ml
10x TBE 4 ml
100% Glycercol 2.8 ml
TEMED 40 µl
10% Ammonium persulfate 400 µl
dH
2
O 24.8 ml
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