Instruction manual

In constant denaturing gels, only a single denaturing condition is used in the gel. With the
constant denaturing gels, it is possible to run more samples under the optimal conditions. Gel
combs are used to form wells in the gel and depending on the number of samples being run,
different combs with different numbers of wells can be used. Constant denaturing gels usually
take about 2–4 hours to reach good resolution between mutant and wild-type DNA fragments.
An example of a constant denaturing gel is shown in Figure 3.6.
In CDGE, separation is time dependent for optimal separation, because wild-type and
mutant fragments immediately begin to partially melt and migrate at a constant rate through
the constant denaturant. Therefore, the running time needed will depend on the resolution
required. A wide separation between wild-type and mutant DNA can be achieved by longer
run times.
Fig. 3.6. Constant denaturing gel. Amplified mutant and wild-type alleles of exon 8 from the TP53
gene. Separation by CDGE run at 130 V for 2.5 hours on a 10% acrylamide gel in 48% denaturant at
56 °C. Lanes 1 and 5 mutant alleles, lanes 2 and 6 wild-type alleles, lanes 3 and 7 mutant and wild-type
alleles, and lanes 4 and 8 homoduplexes and heteroduplexes of the mutant and wild-type alleles.
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