Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 33)

1 2 3
Fig. 4.15. Constant denaturing gel. Amplified mutant and wild-type alleles of exon 8 from the p53 gene.
Separation by CDGE run at 130 V for 2.5 hours on a 10% acrylamide gel in 51% denaturant at 56 °C. Lane 1, mutant
allele; lane 2, wild-type allele; lane 3, mutant and wild-type allele.
Reagent Preparation
The concentration of denaturant is determined from a perpendicular or parallel DGGE
gel. The concentration of acrylamide may vary, depending on the size of the fragment that is
being analyzed. Both 0% and 100% denaturant should be made as stock solutions. A 100%
denaturant is a mixture of 7 M urea and 40% deionized formamide. Reagents for casting and
running CDGE gels are included in the DCode electrophoresis reagent kit for DGGE/CDGE,
catalog number 170-9032.
For different percent crosslinking, use the equation below to determine the amount of Bis
to add. The example stock solution below is for an acrylamide/bis ratio of 37.5:1.
40% Acrylamide/Bis (37.5:1)
Reagent
Amount
Acrylamide 38.93 g
Bis-acrylamide 1.07 g
dH
2
O to 100.0 ml
Filter through a 0.45 µ filter and store at 4°C.
For different percent crosslinking, use the equation below to determine the amount of Bis
to add. The example stock solution is for an acrylamide/bis ratio of 37.5:1.
Polyacrylamide gels are described by reference to two characteristics:
1) Total monomer concentration (%T)
2) Crosslinking monomer concentration (%C)
%T =
gm acrylamide + g bis-acrylamide
x 100
total volume
%C =
gm bis-acrylamide
x 100
gm acrylamide + g bis-acrylamide
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