Reagent Preparation - BIO RAD DCODE Manual

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 elec-
trophoresis 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
Acrylamide
Bis-acrylamide
dH O
2
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)
gm acrylamide + gm bis-acrylamide
%T =
%C =
gm acrylamide + g bis-acrylamide
The table below provides the percentage acrylamide/bis needed for a particular size range.
Gel Percentage
6%
8%
10%
Total Volume
g bis-acrylamide
36
Amount
38.93 g
1.07 g
to 100.0 ml
x 100
x 100
Base Pair Separation
300–1,000 bp
200–400 bp
100–300 bp