Download Report

4100071B 8/31/98 10:05 AM Page CVR3
CE Oligonucleotide
Analysis Kit
Instruction Manual
Catalog Number
148-4140
For Technical Service Call Your Local Bio-Rad Office
or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)
4100071B 8/31/98 10:05 AM Page i
Table of Contents
Section 1
Introduction................................................................1
Section 2
CE Oligonucleotide Kit Components ....................1
2.1
2.2
2.3
2.4
Capillary Preparation and Use ................................................2
Using the Oligonucleotide Size Standards........................2
Sample Preparation............................................................4
Buffer Preparation..................................................................5
Section 3
Analysis Conditions on the BioFocus System......5
3.1
3.2
3.3
Configuration ........................................................................5
Oligonucleotide Analysis Method ....................................7
Analysis of Multiple Samples............................................7
Section 4
System Shutdown Method......................................8
Section 5
Regenerating a Plugged Capillary......................10
Section 6
Product Information..............................................11
4100071B 8/31/98 10:05 AM Page 1
Section 1
Introduction
The CE Oligonucleotide Analysis Kit is optimized for use on
the BioFocus® capillary electrophoresis system. It provides separations of oligonucleotides from 4 to 40 bases in length, and can
be used to determine the purity of synthetic oligonucleotides with
quantitative estimation of the amounts of product and failure
sequences. Separations are achieved by dynamic sieving capillary
electrophoresis. In this technique, incorporation of a hydrophilic
polymer in the electrophoresis buffer solution results in a molecular sieving of nucleic acids. The polymer type and concentration
have been selected to provide unit resolution of oligonucleotides
in the 4–40 mer range. The polymer-containing buffer is replenished between each analysis, which provides reproducibility of
migration time and peak area. The analysis employs capillaries
coated internally with a novel polymer (polyAAEE) which insures
high-resolution separations over many runs.
Section 2
CE Oligonucleotide Kit Components
The CE Oligonucleotide Analysis Kit contains all the reagents
necessary to perform oligonucleotide separations using the
BioFocus capillary electrophoresis system.
1
4100071B 8/31/98 10:05 AM Page 2
•
BioCAP™ Oligonucleotide Analysis Capillary, 2, coated internally with poly(AAEE), 30 cm x 75 µm ID x 375 µm OD,
with detection window 8 cm from the end, 148-3080
•
CE Oligonucleotide Run Buffer, 50 ml, 148-5026
•
CE Oligonucleotide Standards 8–32 mer, 13 peaks, total 0.5
O.D. units, 148-2014
2.1 Capillary Preparation and Use
The AAEE-coated capillary supplied with this kit is designed
for use with the BioFocus user assembled capillary cartridge.
For information on capillary installation, refer to the instructions
included with the BioFocus cartridge assembly kit, catalog number 148-3050, or BioFocus cartridge, catalog number 148-3052. For
use with this kit, the capillary cartridge should be assembled with
24 cm (total length) of capillary installed.
2.2 Using the CE Oligonucleotide Size
Standards
The CE Oligonucleotide Size Standard is a lyophilized mixture of 13 synthetic single-stranded mixed-sequence oligonucleotides ranging in length from 8 to 32 bases (see Figure 1).
When reconstituted with 100 µl of water, the mixture contains
approximately 14 µg/ml of each sequence in TBE buffer
(25 mM Tris, 25 mM borate, 2 mM EDTA) and a total optical
density of 5 A260 units/ml. This standard is used to check the oper2
4100071B 8/31/98 10:05 AM Page 3
ation of the BioFocus instrument and to confirm performance of
the run buffer and coated capillary. Under the conditions described
below, the standard will exhibit 13 major peaks with detector
response of > 5 mAU at 260 nm (see Figure 2). It can be used
directly by pipetting 50 µl of the solution into a 500 µl sample vial.
To avoid loss of solution which may be trapped on the top of the vial,
centrifuge the standards for 10 seconds in a microcentrifuge prior
to opening. The reconstituted standards should be stored at 4 °C.
8 mer
10 mer
12 mer
14 mer
16 mer
18 mer
20 mer
22 mer
24 mer
26 mer
28 mer
30 mer
32 mer
GACTGACT
GACTGACTGT
GACTGACTGACT
GACTGACTGACTGT
GACTGACTGACTGACT
GACTGACTGACTGACTGT
GACTGACTGACTGACTGACT
GACTGACTGACTGACTGACTGT
GACTGACTGACTGACTGACTGACT
GACTGACTGACTGACTGACTGACTGT
GACTGACTGACTGACTGACTGACTGACT
GACTGACTGACTGACTGACTGACTGACTGT
GACTGACTGACTGACTGACTGACTGACTGACT
Fig. 1. Sequences of the synthetic oligonucleotides in the CE
Oligonucleotide Size Standard.
3
14 mer
0.016
12 mer
0.01
0.008
10 mer
0.012
8 mer
Absorbance at 260 nm
0.014
16 mer
18 mer
20 mer
22 mer
24 mer
26 mer
28 mer
30 mer
32 mer
4100071B 8/31/98 10:05 AM Page 4
0.006
0.004
0.002
0
4
5
6
7
8
9
10
11
12
Fig. 2. Electropherogram of the CE Oligonucleotide Size Standards.
2.3 Sample Preparation
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 may have to be desalted by dialysis or ultrafiltration.
4
4100071B 8/31/98 10:05 AM Page 5
2.4 Buffer Preparation
The CE Oligonucleotide Run Buffer should be brought up to
room temperature before use. All run buffer vials should be filled
to capacity to insure proper contact with the capillary and electrode.
Degassing by centrifugation is highly recommended. After pipetting buffer into vials, centrifuge the vials for at least 3 minutes in
a microcentrifuge immediately before inserting them into the
BioFocus carousels.
Section 3
Analysis Conditions on the
BioFocus System
The cartridge data and instrument configuration are shown in
Table 1, the oligonucleotide analysis method in Table 2, and the
shutdown method in Table 3; these should be entered into the
BioFocus database. For systems with carousel temperature control
select either "Not Set" or set the temperature to 20 °C.
3.1 Configuration
The configuration specified in Table 1 includes sufficient
reagents to do 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.
5
4100071B 8/31/98 10:05 AM Page 6
Table 1. Configuration for Oligonucleotide Analysis
ID: OLIGO
Description: Oligonucleotide Analysis
INLET CAROUSEL POSITIONS
Pos
Type
ID/Description
1
R
OLIGO_PREP
2
R
OLIGO_DIP1
3
R
OLIGO_DIP2
4
R
OLIGO_RUN
5
R
WATER
6
R
NITROGEN
7
S
Oligonucleotide Standard
OUTLET CAROUSEL POSITIONS
Pos
Type
ID/Description
1
W
Waste
2
R
OLIGO_RUN
3
R
WATER
CARTRIDGE DATA
Catalog Number: UAC
Length: 24 cm
Diameter: 75 mm
Use Count: 0
Active
6
Contents
Run buffer
Water
Water
Run buffer
Water
Empty
Vial Size
500 µl
500 µl
500 µl
500 µl
500 µl
Empty
500 µl
Contents
Waste
Run buffer
Water
Vial Size
500 µl
500 µl
500 µl
Serial Number: OLIGO
Coated
4100071B 8/31/98 10:05 AM Page 7
3.2 Oligonucleotide Analysis Method
Preparation Cycles—The 120 second, high pressure preparation
cycle (Prep 1) fills the capillary with fresh run buffer at the beginning of each analysis. The two 0 second cycles (Prep 2 and 3)
dip the capillary and electrode into vials containing deionized
water to rinse their surfaces to prevent buffer carry-over into the
sample vial.
Sample Injection—The best resolution and sensitivity are obtained
using electrophoretic injection. However, in cases where the sample contains excessive amounts of salt, pressure injection may
provide better results. Because of the high 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.
Caution: Operating the capillary above 12 kV may shorten the
capillary lifetime.
3.3 Analysis of Multiple Samples
For analysis of multiple samples, a fresh set of run buffer vials
should be used every 10–20 injections.
7
4100071B 8/31/98 10:05 AM Page 8
Table 2. Oligonucleotide Analysis Method
ID: OLIGO
Description: oligonucleotide method
Prep 1: Pre-Inject from [OLIGO_PREP ] to [Waste
] 120 sec
Prep 2: Pre-Inject from [OLIGO_DIP1 ] to [Waste
] 0 sec
Prep 1: Pre-Inject from [OLIGO_DIP2 ] to [Waste
] 0 sec
Polarity:
Electrophoretic
Injection:
Inject Voltage:
Current Limit:
Run Voltage:
Current Limit:
Inlet buffer:
Outlet buffer:
Cartridge
Temperature:
Run Time:
Detector:
Negative - >Positive
4 seconds
10.00 kV
50.00 mA
12.00 kV
50.00 mA
[OLIGO_RUN
[OLIGO_RUN
]
]
40 degrees Celsius
14.00 min
Single wavelength 260 nm
Channel: 1
Range: 0.0200
Rise Time (sec): 1.0
Lamp(s) remain on at the end of the run group
Section 4
System Shutdown Method
When not in use, do not leave the capillary filled with the CE
Oligonucleotide Run Buffer. Drying of the buffer at the ends of the
8
4100071B 8/31/98 10:05 AM Page 9
capillary may cause the capillary to become plugged. After use,
flush the capillary thoroughly with water and then purge 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
Oligonucleotide Shutdown Method.
Table 3. Oligonucleotide Shutdown Method
ID: END
Description: terminates autosequence
Prep 1: Pre-Inject from [WATER
Prep 2: Pre-Inject from [NITROGEN
] to [Waste
] to [Waste
]
]
180 sec
300 sec
No Injection
Polarity:
Run Voltage:
Current Limit:
Inlet buffer:
Outlet buffer:
Cartridge
Temperature:
Run Time:
Detector:
Negative -> Positive
0.00 kV
0.30 mA
[WATER
]
[WATER
]
20 degrees Celsius
1.00 min
Single wavelength 200 nm
Channel: 1
Range: 0.0200
Rise Time (sec): 1.0
Lamp(s) turned off at the end of the run group
9
4100071B 8/31/98 10:05 AM Page 10
Section 5
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.
1. 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
Diagnostics from the toolbar. In the Pressure Diagnostics window, select High Pressure 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.
2. 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 minutes before purging with water.
10
4100071B 8/31/98 10:05 AM Page 11
Section 6
Product Information
Catalog
Number
148-4140
Product Description
CE Oligonucleotide Analysis Kit
148-5026
CE Oligonucleotide Run Buffer
148-2014
CE Oligonucleotide Size Standard
148-3080
BioCAP Oligonucleotide Analysis Capillary, 2
148-3052
BioFocus User Assembled Cartridge
11
4100071B 8/31/98 10:05 AM Page CVR2
Bio-Rad
Laboratories
Life Science
Group
U.S. (800) 4BIORAD California (510) 741-1000 Australia 02-9914-2800 Austria
(1) 877 89 01 Belgium 09-385 55 11 Canada (905) 712-2771 China (01)2046622
Denmark 39 17 9947 Finland 90 804 2200 France (1) 49 60 68 34 Germany 089
31884-0 India 91-11-461-0103 Italy 02-21609 1 Japan 03-5811-6270 Hong Kong
7893300 The Netherlands 0318-540666 New Zealand 09-443 3099 Singapore (65)
272-9877 Spain (91) 661 70 85 Sweden 46 (0) 735 83 00 Switzerland 01-809 55 55
United Kingdom 0800 181134
SIG 020996
Printed in USA
4100071 Rev B