Bio-Rad Fluorescent Assay Kits - Manual (Page 5)

Section 3
Analysis Conditions on the
BioFocus System
The cartridge data and instrument configuration are shown in
Table 1 and the dsDNA analysis method for use with 24 cm capil-
laries in Table 2. A typical shutdown method is depicted in Table 3.
3.1 Configuration
The configuration specified in Table 1 includes sufficient
reagents for up to 30 analyses and a clean-up of the capillary at the
end of the automation sequence. The reagents must be assigned to
carousel positions with vial holders that can accommodate the
vial size recommended in Table 1.
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2.3 Sample Preparation
In Figure 1, the concentration of the DNA sample is 1.0 µg/ml.
In general, DNA component concentrations should be in the range
of 5 to 1,000 pg/ml. The presence of salt in the sample can reduce
the injection efficiency in electrophoretic injection, resulting in
decreased sensitivity. If the salt concentration of the sample is
known to be greater than 50 mM, or if peak response is much
smaller than expected, the sample should be desalted by dialysis
or ultrafiltration.
2.4 Buffer Preparation for Resolving
Fragments of 100–1,000 Base Pairs
The CE dsDNA run buffer should be brought to room tem-
perature before use. It is recommended to filter 12.0 ml of the run
buffer using a 1 µ m filter (Prep-Disc
® filters, catalog number
343-0004). To prepare the CE-LIF run buffer, add 10.0 µl of the
CE Grade SYBR Green I to 10.0 ml of the filtered run buffer (do
not filter run buffer after the addition of the fluorescent dye).
The run buffer containing SYBR Green I should be stored in the
dark to prevent degradation of the dye. If proper handling is exer-
cised, the run buffer with SYBR Green I should be stable for at least
72 hours. All run buffer vials should be filled to capacity to insure
proper contact with the capillary and electrode. Degassing by
centrifugation is strongly recommended. Pipette the buffer into
500 µl vials and centrifuge for at least 2 minutes in a microcentrifuge
before inserting them into the BioFocus carousels.
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