Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 42)

Fig. 4.22. Melting profile of a 191 bp sequence generated with MacMelt software.
Reagent Preparation
The concentration of acrylamide to use varies for the sample being analyzed on the DCode
system. Therefore, a 40% stock solution containing acrylamide and Bis-acrylamide (Bis)
should be made. Reagents for casting and running TTGE gels are included in the DCode
electrophoresis reagent kit for TTGE, catalog number 170-9171.
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.
Polyacrylamide gels are described by reference to two characteristics:
1) The total monomer concentration (%T)
2) The crosslinking monomer concentration (%C)
%T =
gm acrylamide + gm bis-acrylamide
x 100
Total Volume
%C =
g bis-acrylamide
x 100
gm acrylamide + g bis-acrylamide
The table below provides the percentage acrylamide/bis needed for a particular size range.
Gel Percentage Base Pair Separation
6% 300–1,000 bp
8% 200–400 bp
10% 100–300 bp
38