Bio-Rad
Laboratories
CE dsDNA 1000
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United Kingdom 0800 181134
Life Science
Group
SIG 020996 Printed in USA
Fluorescent Detection and
Analysis Kit
Instruction Manual
Catalog Number
148-4133
4006108 Rev B
For Technical Service Call Your Local Bio-Rad Office
or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)
Table of Contents
Section 1 Introduction ..............................................................1
Section 2 CE dsDNA 1000 Fluorescent Detection and
Kit Components......................................................2
2.1 Capillary Preparation and Use ..............................................2
2.2 Using the CE dsDNA 100 Base Pair Ladder ..................3
2.3 Sample Preparation..........................................................4
2.4 Buffer Preparation for Resolving Fragments
of 100–1,000 Base Pairs........................................................4
Section 3 Analysis Conditions on the
BioFocus
3.1 Configuration......................................................................5
3.2 dsDNA Analysis Method ..................................................6
3.3 Analysis of Multiple Samples............................................7
Section 4 System Shutdown Method......................................8
Section 5 Optimization of Nucleic Acid Separations ........10
5.1 Shortening Analysis Time................................................10
5.2 Increasing Resolution ......................................................10
5.3 Regenerating a Plugged Capillary ..................................11
Section 6 Product Information............................................12
®
System ......................................................5
Section 1
Introduction
The CE dsDNA 1000 Fluorescent Detection and Analysis Kit
is optimized for use with the BioFocus®capillary electrophoresis
laser induced fluorescence LIF2detection system and resolves
double-stranded (ds) DNA fragments ranging in size from 100 to
1,000 base pairs. The kit is useful for a variety of applications
including determination of PCR*yields, optimizing PCR conditions,
and characterizing restriction enzyme digests. High performance
capillary electrophoresis is an ideal approach for nucleic acid
separations, providing rapid analysis, quantitative information,
minimal consumption of sample, and direct detection of resolved
fragments.
High resolution DNA separations are achieved by dynamic
sieving**capillary electrophoresis, a technique that incorporates a
hydrophilic polymer in the electrophoresis buffer . The polymer -containing buffer is replenished between each analysis, providing
reproducibility of migration time and peak area. The analysis
employs BioCap™capillaries which are coated internally with a
novel polymer (polyAAEE), insuring high resolution separations
over many runs.
SYBR®Green I, the fluorescent dye included in the kit, is an
intercalating monomeric dye with the excitation maximum close
to the 488 nm line of the argon ion laser, extremely low intrinsic
fluorescence, and very high affinity for dsDNA.
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Section 2
CE dsDNA 1000 Fluorescent Detection
and Kit Components
The kit contains all the reagents for 80 setups, with 30 runs per
setup using the BioFocus LIF2detection system.
• T wo BioCAP DNA analysis capillaries, 50 cm x 75 µm ID x
375 µm OD
• CE dsDNA run buffer in 2x TBE, 60 ml (2 bottles)
• CE-LIF dsDNA 100 bp ladder, 200 µl at 1.0 µ g/ml in TE
buffer, pH 8.0
• CE Grade SYBR Green I, 200 µ l (Since this fluorescent dye
is light sensitive, do not leave exposed to light when not in
use. Store the fluorescent dye at 4 °C or less.)
• Instruction manual
2.1 Capillary Preparation and Use
The BioCAP DNA analysis capillary supplied with this kit is
designed for use with the BioFocus LIF user assembled capillary
cartridge. For information on capillary installation, refer to the
instructions included with the BioFocus LIF cartridge assembly kit,
catalog number 148-3054. For use with this kit, the capillary cartridge can be assembled with 24 to 44 cm (total length) of capillary installed.
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2.2 Using the CE dsDNA 100 Base Pair
Ladder
The CE-LIF dsDNA 100 bp ladder is a mixture of 10 doublestranded sequences ranging in length from 100 bp to 1,000 bp in exact
100 bp increments. Under the conditions described below, the standard
will exhibit 10 major peaks (see Figure 1). Pipette 20–50 µ l of the
CE-LIF dsDNA 100 bp ladder into a 500 µl sample vial. Routinely, the
CE-LIF 100 bp ladder should be stored at 4 °C. However, longer term
storage at -20 °C may increase shelf life.
0.9
0.8
0.7
0.6
0.5
0.4
RFU
0.3
0.2
0.1
0
-0.1
Fig. 1. Separation of 100 bp ladder.
100 bp
200 bp
300 bp
400 bp
12 16 20 24
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Minutes
500 bp
600 bp
3
700 bp
800 bp
900 bp
1,000 bp
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 temperature 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 exercised, 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.
Section 3
Analysis Conditions on the
BioFocus System
The cartridge data and instrument configuration are shown in
T able 1 and the dsDNA analysis method for use with 24 cm capillaries 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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5
Table 1. BioFocus Configuration for dsDNA Analysis
ID: DNA Description: dsDNA Analysis
INLET CAROUSEL POSITIONS Amount
Pos Type ID/Description Contents Vial Size in Vial
1 R DNA_RUN Run buffer 500 µl 500 µl
2 R DNA_PREP Run buffer 500 µl 500 µl
3 R WATER_DIP Water 500 µl 500 µl
4 R WATER_DIP Water 500 µl 500 µl
9 R WATER/Shutdown Water 500 µl 500 µl
10 R NITROGEN/Shutdown Empty 500 µl
11 S 100 bp ladder Sample 500 µl 20–50 µl
OUTLET CAROUSEL POSITIONS Amount
Pos Type ID/Description Contents Vial Size in Vial
1 R DNA_RUN Run buffer 500 µl 500 µl
2 R WATER/Shutdown Water 500 µl 500 µl
32 W WASTE Water (100 µl) 500 µl 100 µl
CARTRIDGE DATA
Catalog Number: UAC Serial Number: DNA
Length: 24 cm Diameter: 75 mm Coated
Use Count: 0 Active
3.2 dsDNA Analysis Method
Preparation Cycles—The 45 second, high pressure preparation cycle
(Prep cycle 1) fills the capillary with fresh run buffer at the beginning of each analysis. The 0 second cycles (Prep cycle 2 and 3) dip
the capillary and electrode into vials containing deionized water to
rinse their surfaces and prevent buffer carry-over into the sample
vial (see Table 2).
Sample Injection—The best resolution and sensitivity are normally
obtained using electrophoretic injection at constant voltage and a
current limit of 100 µ A. However, in cases where the sample
contains excessive amounts of salt, pressure injection may
provide better results. Because of the viscosity of the run buffer,
a pressure injection value of 100 psi*sec or more should be used;
this injects a volume of approximately 15 nl.
3.3 Analysis of Multiple Samples
For analysis of multiple samples, a fresh set of run buffer vials
should be used every 30 injections.
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7
Table 2. dsDNA Analysis Method for 24 cm Capillary
Set the carousel temperature to 20 °C
Section 4
System Shutdown Method
Drying of the buffer at the ends of the capillary may cause
the capillary to become plugged. For short term storage (24 hours),
leave the capillary filled with the CE dsDNA run buffer and place
the capillary ends in test tubes filled with deionized water at 4 °C.
For long term storage, the capillary should be flushed thoroughly
with water and then purged with nitrogen. This may be programmed
to run automatically at the end of an automation sequence using the
method parameters shown in Table 3. The Shutdown Method
included in the BioFocus software can be used as a template to
quickly program the dsDNA Shutdown Method.
Table 3. dsDNA Shutdown Method
ID: END Description: terminates autosequence
Prep 1: Pre-Inject from [WATER ] to [Waste ] 180 sec
Prep 2: Pre-Inject from [NITROGEN ] to [Waste ] 300 sec
No Injection
Polarity: Negative -> Positive
Run Voltage: 0.00 kV
Current Limit: 0.30 mA
Inlet buffer: [WATER ]
Outlet buffer: [WATER ]
Cartridge
Temperature: 20 degrees Celsius
Run Time: 1.00 min
Detector: LIF
Channel: 1
Laser(s) turned off at the end of the run group
8
9
Section 5
Optimization of Nucleic Acid
Separations
5.3 Regenerating a Plugged Capillary
If the capillary becomes plugged (as evidenced by zero current, or failure of the liquid to appear at the capillary outlet during
manual purging), there are two ways to unplug the capillary.
5.1 Shortening Analysis Time
The time of analysis can be shortened by increasing the run
voltage, although this can result in some loss in performance. For
example, running at 4.0 kV on a 24 cm capillary can shorten the
analysis time of the CE-LIF dsDNA 100 bp ladder to about
12 minutes.
5.2 Increasing Resolution
Resolution can be improved by increasing the length of the
capillary, but this will also result in longer analysis times. To maintain the same field strength, the operating voltage should be
increased proportionately to the increase in capillary length.
Resolution also depends upon the width of the sample zone
loaded into the capillary; narrow zones improve resolution. Narrow
sample zones can be obtained by reducing the injection time and/or
the injection voltage for electrophoretic injection, or the psi*second
value for pressure injection.
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On-line method
Leave the capillary cartridge installed in the BioFocus
system. Place vials containing 100 µl deionized water in the inlet
and outlet carousel. Select Pressure Diagnosticsfrom the toolbar.
In the Pressure Diagnostics window , select High Pr essur e Mode,
select the carousel positions containing the water vials, enter
180 seconds for the Maximum Limit for Testing, then press
Start. After the 3 minute purge period, exit from Pressure
Diagnostics and visually check the liquid level in the inlet water
vial. If the vial is empty, the capillary has been unplugged.
Off-line method
Remove the capillary cartridge from the instrument and
immerse the capillary ends in hot (70 °C, not boiling) deionized
water for 10–15 minutes and check by manually purging with
water. Alternatively, immerse the capillary ends in a sonic bath
filled with deionized water and sonicate for about 5–10 minute
before purging with water.
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Section 6
Product Information
Catalog
Number Product Description
148-4133 CE dsDNA 1000 Fluorescent Detection and
Analysis Kit
148-5041 CE dsDNA Run Buffer
148-2021 CE-LIF ds DNA 100 bp ladder
148-5100 CE Grade SYBR Green I Fluorescent Dye
148-3084 BioCAP DNA Analysis Capillary, 2
148-3054 BioFocus LIF User Assembled Cartridge
* PCR is covered by U.S. patents owned by F. Hoffmann-La Roche & Co.
** Dynamic sieving CE is covered by U.S. patent 5,089,111 issued to Bio-Rad Laboratories.
SYBR Green I is a registered trademark of Molecular Probes, Inc., (M.P.I.) and SYBR Green I is
licensed from M.P.I. for CE research.
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