Beckman Coulter CEQ 8000, User Manual

Page: 248 / 382

Fragment Analysis Procedures
234 CEQ™ 8000 Genetic Analysis System
• For example, in the SizeStandard-400, the peaks in the larger half of the
size range are spaced 20 nucleotides apart, while some in the smaller half
of the size range are spaced 10 nucleotides apart.
Setting the Analysis Method
1. Select the standard to be run with the sample from the
Size Standard
drop-down
list box.
• The size standards available are:
SizeStandard-400
,
SizeStandard-600
, &
SizeStandard-80
.
• Your selection in this list box will make available various size standards to
be selected in the
Standard Fragments
frame to the right of the
window.
• To use all fragments in the selected standard, click the
Select All
button.
• To select individual fragments in the selected standard, select the check
box next to each of the fragment sizes you want to run with the sample.
• If no standard is to be run with the sample, click the
Deselect All
button.
If you do not use a size standard when analyzing the data, the
unknown fragments will not be sized.
2. The
Dye
field displays the dye label on the size standard fragments.
3. The
Size Standard Fit Coefficient
field displays the standard deviation of the
differences between the actual fragment sizes and the apparent sizes of the
standard fragments determined from a large number of repeated runs. This
parameter is sometimes called the “lack-of-fit”.
4. Select the empirical model that will be used to generate the curve that best fits the
identified standard fragments from the
Model
drop-down list box.
The relationship between fragment size and mobility (or migration time) in
CE with replaceable gels is non-linear, and is not described with sufficient
accuracy with any theoretical model to yield reliable fragment size
estimates. However, polynomials of varying degrees serve as practical
empirical models that will accurately account for this non-linearity.
The model applied to the calibration curve can be either linear, quadratic, cubic,
quartic or Local Southern, depending on the standard used.
• A
Linear Fit
will result in a straight line model which will only be useful for a set
of size standards spanning a very small range.
• A
Quadratic Fit
will adequately account for the non-linear dependence of
mobility on size under some conditions, but usually over relatively short size
ranges which are well separated from the size limits of the separation system.
• Generally, a
Cubic Curve
will represent the curvature in a time or mobility
versus size plot well enough that errors in estimated sizes are dominated by other
factors, e.g., effects of primary structure on mobility.
This is usually the best
choice for routine use with the SizeStandard-400 set .