High performance capillary electrophoresis - T.E.A.M.

Modes
a)
ds 500 base pairs
This sample contains a ladder of 1-kbp DNA extending from
1 to 12-kbp and numerous smaller fragments which result
from the enzymatic nature of the sample preparation. The
resolution of fragments from 75 bp to 12 kbp in a single
analysis illustrates the wide sample range of CGE.
b)
10 20 30
ss 500 bases
10 20 30
ds 500 bp
An example of PCR product analysis is shown in figure 44.
Here, the analysis of a single-stranded DNA prepared by
asymmetric PCR is shown. The peaks were identified by a
calibration curve obtained using DNA size standards. Note
that the single-stranded DNA migrates slower than the
double-stranded DNA of the same size due to increased
random three-dimensional structure. In addition, the lack of
specificity of asymmetric PCR yields more by-products than
normal PCR.
Figure 44
PCR analysis of single and double
stranded DNA
Conditions: Uncrosslinked polyacrylamide
(9 % T, 0 % C), 100 mM Trisborate,
pH 8.3, E = 300 V/cm,
i = 9 mA, l = 20 cm, L = 40 cm,
id = 75 mm, l = 260 nm, polyacrylmide
coated capillary
In the area of protein separations, much research has gone
into SDS-gels for size-based separation. Both standard SDS-
PAGE gels and linear gels with SDS have been employed.
The separation of size standards is shown in figure 45 using
crosslinked polyacrylamide. Alternatively, linear dextran
polymers have been used since these offer higher gel
stability and lower background absorbance at low wavelengths.
1 Tracking dye
2 Lysozyme
3 β-lactoglobulin
4 Trypsinogen
5 Pepsin
6 Egg albumin
7 Bovine albumin
1
2
3
4
5
6
7
Figure 45
SDS-PAGE separation of protein
standards 23
Conditions: Bis-crosslinked polyacrylamide
(7.5 % T, 5 % C), 100 mM trisborate,
0.1 % SDS, 8 M urea, pH
7.3, E = 300 V/cm, i = 12 mA,
l = 15 cm, id = 75 mm, l = 280 nm,
polyacrylamide coated capillary
12 18 25
74