Big Dye XTerminator Purification Kit

Big Dye XTerminator Purification Kit
Applied Biosystems
BigDye® XTerminator™
Purification Kit
Protocol
© Copyright 2007, Applied Biosystems. All rights reserved.
Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that
may appear in this document.
APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR
IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR
PURPOSE. IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY,
OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE,
MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT,
INCLUDING BUT NOT LIMITED TO THE USE THEREOF.
For Research Use Only. Not for use in diagnostic procedures.
NOTICE TO PURCHASER: LICENSE DISCLAIMER
This product is compatible with DNA sequencing or fragment analysis methods covered by patents owned or licensed by Applied
Biosystems. No license under these patents to use these methods is conveyed expressly, by implication, or by estoppel. A license to
use the DNA sequencing or fragment analysis methods for certain research and development activities is available from Applied
Biosystems. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied
Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
TRADEMARKS:
Applera, Applied Biosystems, AB (Design), ABI PRISM, and BigDye are registered trademarks and Hi-Di, MicroAmp, POP-4,
POP-6, POP-7, SAM, and XTerminator are trademarks of Applera Corporation or its subsidiaries in the US and/or certain other
countries.
All other trademarks are the sole property of their respective owners.
Part Number 4374408 Rev. C
07/2007
Contents
1
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
How to Obtain More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x
How to Obtain Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x
BigDye® XTerminator™ Purification Kit Protocol . . . . . . . . 1
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Before You Begin Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Performing Purification – Sequential Pipetting . . . . . . . . . . . . . . . . . . . . 15
Performing Purification – Premix Pipetting . . . . . . . . . . . . . . . . . . . . . . . 20
Storage of Purified Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Appendix A: Run Modules . . . . . . . . . . . . . . . . . . . . . . . 37
Appendix B: DNA Quantity Guidelines . . . . . . . . . . . . . . 41
Appendix C: Plate Sealing Procedure . . . . . . . . . . . . . . 43
Appendix D: Mixing Guidelines . . . . . . . . . . . . . . . . . . . . 47
Mixing with the Scientific Industries Digital Vortex-Genie 2 . . . . . . . . . . 47
Mixing with the Union Scientific Vertical Shaker . . . . . . . . . . . . . . . . . . . 50
Mixing with the IKA MS 3 Digital Vortexer . . . . . . . . . . . . . . . . . . . . . . . 52
Mixing with the Taitec MicroMixer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Appendix E: Performing Purification – Robotic Pipetting . .
57
BigDye® XTerminator™ Purification Kit Protocol
iii
Contents
iv
BigDye® XTerminator™ Purification Kit Protocol
Preface
This preface covers:
Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
How to Obtain More Information . . . . . . . . . . . . . . . . . . . . . . . . . . x
How to Obtain Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x
Safety
Safety Alert
Words
Four safety alert words appear in Applied Biosystems user
documentation at points in the document where you need to be aware
of relevant hazards. Each alert word—IMPORTANT, CAUTION,
WARNING, DANGER—implies a particular level of observation or
action, as defined below.
Definitions
IMPORTANT! – Indicates information that is necessary for proper
instrument operation, accurate chemistry kit use, or safe use of a
chemical.
– Indicates a potentially hazardous situation that,
if not avoided, may result in minor or moderate injury. It may also be
used to alert against unsafe practices.
– Indicates a potentially hazardous situation that,
if not avoided, could result in death or serious injury.
– Indicates an imminently hazardous situation
that, if not avoided, will result in death or serious injury. This signal
word is to be limited to the most extreme situations.
BigDye® XTerminator™ Purification Kit Protocol
v
Preface
Chemical Hazard
Warning
Chemical Safety
Guidelines
About MSDSs
CHEMICAL HAZARD. Some of the chemicals
used with Applied Biosystems instruments and protocols are
potentially hazardous and can cause injury, illness, or death.
To minimize the hazards of chemicals:
• Read and understand the Material Safety Data Sheets (MSDSs)
provided by the chemical manufacturer before you store, handle,
or work with any chemicals or hazardous materials. (See “About
MSDSs,” on page vi.)
• Minimize contact with chemicals. Wear appropriate personal
protective equipment when handling chemicals (for example,
safety glasses, gloves, or protective clothing). For additional
safety guidelines, consult the MSDS.
• Minimize the inhalation of chemicals. Do not leave chemical
containers open. Use only with adequate ventilation (for
example, fume hood). For additional safety guidelines, consult
the MSDS.
• Check regularly for chemical leaks or spills. If a leak or spill
occurs, follow the manufacturer’s cleanup procedures as
recommended in the MSDS.
• Comply with all local, state/provincial, or national laws and
regulations related to chemical storage, handling, and disposal.
Chemical manufacturers supply current Material Safety Data Sheets
(MSDSs) with shipments of hazardous chemicals to new customers.
They also provide MSDSs with the first shipment of a hazardous
chemical to a customer after an MSDS has been updated. MSDSs
provide the safety information you need to store, handle, transport,
and dispose of the chemicals safely.
Each time you receive a new MSDS packaged with a hazardous
chemical, be sure to replace the appropriate MSDS in your files.
vi
BigDye® XTerminator™ Purification Kit Protocol
Safety
Obtaining
MSDSs
The MSDSs for many chemicals supplied by Applied Biosystems are
available to you free 24 hours a day. To obtain MSDSs:
1. Go to https://docs.appliedbiosystems.com/msdssearch.html
2. In the Search field of the MSDS Search page:
a. Type in the chemical name, part number, or other
information that you expect to appear in the MSDS of
interest.
b. Select the language of your choice.
c. Click Search.
3. To view, download, or print the document of interest:
a. Right-click the document title.
b. Select:
• Open – To view the document
• Save Target As – To download a PDF version of the
document to a destination that you choose
• Print Target – To print the document
4. To have a copy of an MSDS sent by fax or e-mail, in the Search
Results page:
a. Select Fax or Email below the document title.
b. Click RETRIEVE DOCUMENTS at the end of the
document list.
c. Enter the required information.
d. Click View/Deliver Selected Documents Now.
Note: For the MSDSs of chemicals not distributed by Applied
Biosystems, contact the chemical manufacturer.
BigDye® XTerminator™ Purification Kit Protocol
vii
Preface
Chemical Waste
Hazards
HAZARDOUS WASTE. Refer to Material
Safety Data Sheets and local regulations for handling and disposal.
CHEMICAL WASTE HAZARD. Wastes
produced by Applied Biosystems instruments are potentially
hazardous and can cause injury, illness, or death.
CHEMICAL STORAGE HAZARD. Never
collect or store waste in a glass container because of the risk of
breaking or shattering. Reagent and waste bottles can crack and leak.
Each waste bottle should be secured in a low-density polyethylene
safety container with the cover fastened and the handles locked in the
upright position. Wear appropriate eyewear, clothing, and gloves
when handling reagent and waste bottles.
Chemical Waste
Safety Guidelines
viii
To minimize the hazards of chemical waste:
• Read and understand the Material Safety Data Sheets (MSDSs)
provided by the manufacturers of the chemicals in the waste
container before you store, handle, or dispose of chemical waste.
• Provide primary and secondary waste containers. (A primary
waste container holds the immediate waste. A secondary
container contains spills or leaks from the primary container.
Both containers must be compatible with the waste material and
meet federal, state, and local requirements for container
storage.)
• Minimize contact with chemicals. Wear appropriate personal
protective equipment when handling chemicals (for example,
safety glasses, gloves, or protective clothing). For additional
safety guidelines, consult the MSDS.
• Minimize the inhalation of chemicals. Do not leave chemical
containers open. Use only with adequate ventilation (for
example, fume hood). For additional safety guidelines, consult
the MSDS.
• Handle chemical wastes in a fume hood.
• After emptying the waste container, seal it with the cap
provided.
• Dispose of the contents of the waste tray and waste bottle in
accordance with good laboratory practices and local,
state/provincial, or national environmental and health
regulations.
BigDye® XTerminator™ Purification Kit Protocol
Safety
Waste Disposal
If potentially hazardous waste is generated when you operate the
instrument, you must:
• Characterize (by analysis if necessary) the waste generated by
the particular applications, reagents, and substrates used in your
laboratory.
• Ensure the health and safety of all personnel in your laboratory.
• Ensure that the instrument waste is stored, transferred,
transported, and disposed of according to all local,
state/provincial, and/or national regulations.
IMPORTANT! Radioactive or biohazardous materials may require
special handling, and disposal limitations may apply.
Biological Hazard
Safety
BIOHAZARD. Biological samples such as
tissues, body fluids, infectious agents, and blood of humans and other
animals have the potential to transmit infectious diseases. Follow all
applicable local, state/provincial, and/or national regulations. Wear
appropriate protective equipment, which includes but is not limited
to: protective eyewear, face shield, clothing/lab coat, and gloves. All
work should be conducted in properly equipped facilities using the
appropriate safety equipment (for example, physical containment
devices). Individuals should be trained according to applicable
regulatory and company/institution requirements before working
with potentially infectious materials. Read and follow the applicable
guidelines and/or regulatory requirements in the following:
• U.S. Department of Health and Human Services guidelines
published in Biosafety in Microbiological and Biomedical
Laboratories
(stock no. 017-040-00547-4; http://bmbl.od.nih.gov)
• Occupational Safety and Health Standards, Bloodborne
Pathogens (29 CFR§1910.1030; http://www.access.gpo.gov/
nara/cfr/waisidx_01/ 29cfr1910a_01.html).
• Your company’s/institution’s Biosafety Program protocols for
working with/handling potentially infectious materials.
Additional information about biohazard guidelines is available at:
http://www.cdc.gov
BigDye® XTerminator™ Purification Kit Protocol
ix
Preface
How to Obtain More Information
Related
Documentation
The BigDye® XTerminator™ Purification Kit Product Insert, which
is shipped with the kit, summarizes how to install BigDye®
XTerminator™ Kit run modules and perform the purification
protocol. Additional instrument-specific documents are listed in
Table 3 on page 8.
Additional information is available at the BigDye XTerminator web
page at:
www.appliedbiosystems.com/gameoverblobs
Send Us Your
Comments
Applied Biosystems welcomes your comments and suggestions for
improving its user documents. You can e-mail your comments to:
[email protected]
IMPORTANT! The e-mail address above is for submitting comments
and suggestions relating only to documentation. To order documents,
download PDF files, or for help with a technical question, go to
www.appliedbiosystems.com, then click the link for Support. (See
“How to Obtain Support” (x) below).
How to Obtain Support
For the latest services and support information for all locations, go to
www.appliedbiosystems.com, then click the link for Support.
At the Support page, you can:
• Search through frequently asked questions (FAQs)
• Submit a question directly to Technical Support
• Order Applied Biosystems user documents, MSDSs, certificates
of analysis, and other related documents
• Download PDF documents
• Obtain information about customer training
• Download software updates and patches
In addition, the Support page provides access to worldwide telephone
and fax numbers to contact Applied Biosystems Technical Support
and Sales facilities.
x
BigDye® XTerminator™ Purification Kit Protocol
BigDye® XTerminator™ Purification Kit
Protocol
Overview
The BigDye® XTerminator™ Purification Kit is designed to sequester
cycle-sequencing reaction components such as salt ions,
unincorporated dye terminators, and dNTPs, to prevent their coinjection with dye-labeled extension products.
BigDye® XTerminator™ Solutions are added directly to finished
sequencing reactions and vortexed. During vortexing, the BigDye
XTerminator reagents capture and immobilize unwanted
components. After vortexing, the reactions are briefly centrifuged to
move the insoluble fraction of the reagent mixture and the captured
reaction components to the bottom of the reaction well. The purified
dye-labeled extension products remain in the supernatant and are
injected directly from the supernatant into the DNA sequencer, where
they are analyzed using specialized BigDye XTerminator run
modules.
Supported
Injection
Configurations
Table 1 shows the instrument, plate, and method of sealing that allow
for direct injection of samples purified with the BigDye XTerminator
Purification Kit.
Table 1 Supported software, seals, and injection configurations for the BigDye®
XTerminator™ Purification Kit
Plate Type and Reaction
Volume/Well
Seals Supported for
Direct Injection
Direct Injection
Supported ‡
Run Module
ABI PRISM® 3100/3100-Avant Genetic Analyzer with Data Collection Software v 2.0
384-well, 5-µL
Septa
No
Standard
96-well, 10-µL
Septa
Yes
BDx
96-well, 20-µL
Septa
Yes
BDx
BigDye® XTerminator™ Purification Kit Protocol
1
Overview
Table 1 Supported software, seals, and injection configurations for the BigDye®
XTerminator™ Purification Kit (continued)
Plate Type and Reaction
Volume/Well
Seals Supported for
Direct Injection
Direct Injection
Supported ‡
Run Module
ABI PRISM® 3100/3100-Avant Genetic Analyzer with Data Collection Software v1.x
384-well, 5-µL
Septa
No
Standard
96-well, 10-µL
Septa
No
Standard
96-well, 20-µL
Septa
No
Standard
Applied Biosystems 3130/3130xl Genetic Analyzer with Data Collection Software v3
384-well, 5-µL
Septa
No
Standard
96-well, 10-µL
Septa
Yes
BDx
96-well, 20-µL
Septa
Yes
BDx
Applied Biosystems 3730/3730xl DNA Analyzer with Data Collection Software v2 or v3
384-well, 5-µL
Heat seal
Yes
BDx
Septa
No
Standard
96-well, 10-µL
Heat seal or septa
Yes
BDx
96-well, 20-µL
Heat seal or septa
Yes
BDx
384-well, 5-µL
Septa
No
Standard
96-well, 10-µL
Septa
No
Standard
96-well, 20-µL
Septa
No
Standard
ABI PRISM® 310 Genetic Analyzer
Applied Biosystems 3700 DNA Analyzer
384-well, 5-µL
Septa or heat seal
No
Standard
96-well, 10-µL
Septa or heat seal
No
Standard
96-well, 20-µL
Septa or heat seal
No
Standard
‡ For configurations where direct injection is not supported, transfer the supernatant to a new plate.
2
BigDye® XTerminator™ Purification Kit Protocol
Overview
Note: MicroAmp™ Fast 96-Well Reaction Plates (PN 4346907) can
be used with the BigDye XTerminator Purification Kit for
sequencing reaction volumes 10 µL or less.
About the BigDye
XTerminator
Purification Kit
Kit Size
The BigDye XTerminator Purification Kit has two reagents:
• XTerminator Solution – Captures unincorporated dye
terminators and free salts from the post cycle-sequencing
reaction.
• SAM™ Solution – Improves BigDye XTerminator reagent
performance and stabilizes the sample after purification.
Approximate
Number of 20-µL
Reactions
Volume of Each Kit Reagent (mL)
XTerminator™
Solution
Part Number
SAM™ Solution
2-mL
100
2
9
4376486
20-mL
1,000
20
90
4376487
50-mL
2,500
50
225
4376484
800-mL
40,000
800
3,600
4376485
Handling and Storage
If you plan to work with the BigDye XTerminator Purification Kit
reagents for more than 30 minutes, keep them on ice with the caps
closed between pipetting steps. Avoid freezing the reagents.
When stored at 4 °C, XTerminator Solution is stable through the
expiration date on the package and bottle label.
When stored at room temperature or 4 °C, SAM Solution is stable
through the date on the package and bottle label. Storage at room
temperature may decrease the likelihood of precipitation.
BigDye® XTerminator™ Purification Kit Protocol
3
Overview
About This
Protocol
4
This protocol describes:
• Required and recommended materials for using the BigDye
XTerminator Purification Kit.
• Instructions for preparing for and performing the purification
protocol.
• Instructions for installing and updating run modules for the Data
Collection software.
BigDye® XTerminator™ Purification Kit Protocol
Overview
Sequencing
Workflow
Figure 1 shows how using the BigDye XTerminator Purification Kit
fits into a sequencing workflow.
Perform cycle sequencing
GeneAm
PCR Systemp ®
9700
Genotyping
PCR System
9600
Thermal cyclers
1
2
3
4
5
6
7
8
9
10
11
12
Extension Product Purification
A
Add BigDye® XTerminator™
reagents to reaction plate
B
C
D
E
F
G
H
96-well plate
384-well plate
Vortex reaction plate
Vortexer
G R 2170
M ic ro F lu id ic C a rd
S o rv a ll C e n trifu g e
Centrifuge plate
Centrifuge
Select appropriate run module in
Data Collection software
Perform electrophoresis
3730/3130/3100 instruments
Figure 1
Sequencing workflow
BigDye® XTerminator™ Purification Kit Protocol
5
Materials
For details about dye terminators, cycle sequencing, and
electrophoresis, refer to the appropriate document or instrument user
guide (see Table 3 on page 8).
Materials
Applied
Biosystems
Instruments
Table 2
Instruments and accessories from Applied Biosystems
Source or Part
Number
Item
One of the following Applied Biosystems DNA or
Genetic Analyzers and corresponding data
collection software:
• 3730xl DNA Analyzer with Data Collection
Software v2.0 or 3.0
• 3730 DNA Analyzer with Data Collection
Software v2.0 or 3.0
• 3130xl Genetic Analyzer with 3130 Data
Collection Software v3.0
• 3130 Genetic Analyzer with 3130 Data
Collection Software v3.0
• ABI PRISM® 3100 Genetic Analyzer with
3100 Data Collection Software v2.0
• ABI PRISM® 3100-Avant Genetic Analyzer with
3100-Avant Data Collection Software v2.0
Either of the following sequencing kits, as
necessary:
• BigDye® Terminator v3.1 Cycle Sequencing Kit
• BigDye® Terminator v1.1 Cycle Sequencing Kit
6
Contact your
Applied Biosystems
sales
representative.
Contact your
Applied Biosystems
sales
representative.
BigDye® XTerminator™ Purification Kit Protocol
Materials
Table 2
Instruments and accessories from Applied Biosystems
Item
MicroAmp ™ Fast 96-Well Reaction Plates
(0.1-mL) (10 plates).
Source or Part
Number
4346907
MicroAmp ™ Fast Optical 96-Well Reaction Plates
with Barcode (0.1-mL). Available quantities are:
• 20
• 200
4346906
4366932
MicroAmp ™ Optical 96-Well Reaction Plates.
Available quantities are:
• 20
• 500
N8010560
4316813
MicroAmp ™ Optical 96-Well Reaction Plates with
Barcode. Available quantities are:
• 20
• 500
4306737
4326659
MicroAmp ™ Optical 384-Well Reaction Plates
with Barcode. Available quantities are:
• 50
• 500
• 1000
4309849
4326270
4343814
MicroAmp ™ Fast Optical 48-Well Reaction Plates
(20 plates)
4375816
Plate accessories, such as:
•
•
•
•
•
•
MicroAmp™ Clear Adhesive Films (100)
MicroAmp™ Adhesive Film Applicator
MicroAmp ™ Multi Removal Tool
96-Well Plate Septa (20)
MicroAmp™ Splash Free 96-Well Base
Heat Seal Film for Sequencing and Fragment
Analysis Sample Plates
BigDye® XTerminator™ Purification Kit Protocol
4306311
4333183
4313950
4315933
4312063
4337570
7
Materials
Table 3
Applied Biosystems documents
Item
Part Number
One or more of the following, as applicable:
Vortexers and
Accessories
• Applied Biosystems 3730/3730xl DNA
Analyzers Sequencing Chemistry Guide
• Applied Biosystems 3730/3730xl DNA
Analyzer Getting Started Guide
• Applied Biosystems 3130/3130xl Genetic
Analyzers Getting Started Guide
• ABI PRISM® 3100/3100-Avant Genetic
Analyzer User Guide
4331467
BigDye® Terminator v3.1 Cycle Sequencing Kit
Protocol
4337035
BigDye® Terminator v1.1 Cycle Sequencing Kit
Protocol
4337036
4359476
4352715
4347102
The vortexing step of the BigDye XTerminator Purification Kit
protocol is critical to achieving optimum performance. The use of
one of the vortexers below is strongly recommended.
Select a vortexer and the indicated accessories based on the type of
plates you plan to use and the number of plates you want to load at
the same time:
• 96-well plates – Table 4
• 384-well plates – Table 5
The Digital Vortex-Genie 2, the IKA MS3 Digital Vortexer, the IKA
Works 3 Vortexer, and the MixMate can vortex one plate at a time.
The Taitec MicroMixer E-36 can vortex two plates at a time. The
Union Scientific Vertical Shaker can accommodate up to six 96-well
plates or eighteen 384-well plates at once.
If you select a vortexer that is not included in Table 4 or 5, best
results are obtained from a vortexer with a minimum of 2000 rpm and
a maximum orbital diameter of 4 mm.
Note: Additional supported vortexers are listed at the BigDye
XTerminator web page at:
www.appliedbiosystems.com/gameoverblobs
8
BigDye® XTerminator™ Purification Kit Protocol
Materials
Click the Product Description tab and look for the links in step 4
under the heading “Simple Purification Process”.
Table 4
Vortexers and adapters for 96-well plates
Vortexer and Source
Digital Vortex-Genie 2, Applied
Biosystems, 120 V, 60 Hz (PN SI-A536)
Digital Vortex-Genie 2, Applied
Biosystems, 100 V, 50/60 Hz (PN SI-A586)
Digital Vortex-Genie 2, Applied
Biosystems, 230 V, 50 Hz, with:
•
•
•
•
No Plug (PN SI-A546)
European Plug (PN SI-A556)
British Plug (PN SI-A566)
Swiss Plug (PN SI-A576)
All models above include shock-absorbing
feet, Microplate Adapter for Applied
Biosystems, and elastic bands.
Scientific Industries, Inc.
70 Orville Drive
Bohemia, NY 11716
Tel: (631) 567-4700
Fax: (631) 567-5896
Adapter or Accessories
If you have another model
of Digital Vortex-Genie 2,
you need:
• Shock-absorbing feet
(PN 0K-0400-900)
• Microplate adapter for
Applied Biosystems
(includes 2 elastic
bands) (PN SI-0513)
• Replacement elastic
bands (2) for Microplate
adapter for Applied
Biosystems
(PN 0K-0513-900)
If you use the Recessed
Platform
(PN 504-0039-00):
• Elastic bands
(PN 568-0001-00)
[email protected]
www.scientificindustries.com
IKA MS 3 Digital (PN 3319000)
IKA® Works, Inc.
2635 North Chase Pkwy SE
Wilmington, NC 28405-7419
This vortexer includes the MS 3.4 Microtiter
Attachment (PN 3426400).
Tel: (910) 452-7059
Fax: (910) 452-7693
From any office supply
vendor:
• Elastic bands, size 64
(90 mm ! 6 mm)
Note: For regions outside
North America, an
electrical plug adapter
may be required.
[email protected]
www.ika.net
BigDye® XTerminator™ Purification Kit Protocol
9
Materials
Table 4
Vortexers and adapters for 96-well plates (continued)
Vortexer and Source
Adapter or Accessories
From IKA Works:
IKA Vortex 3 (PN 3340001)
®
IKA Works, Inc.
2635 North Chase Pkwy SE
Wilmington, NC 28405-7419
• VG 3.3 Universal
attachment
(Ident. No. 3342400)
Tel: (910) 452-7059
Fax: (910) 452-7693
From any office supply
vendor:
[email protected]
www.ika.net
• Elastic band, size 117B
(3 mm ! 178 mm)
Taitec MicroMixer E-36 (PN BNE36)
From Applied Biosystems:
Distributed in the US by:
Bionexus Inc.
222 Madison Street, Suite 200
Oakland, CA 94607
• MicroAmp™ 96-Well
Base (PN N8010531)
Tel: (510)-625-8400
Fax: 510-625-8419
[email protected]
www.bionexus.net
Union Scientific Vertical Shaker
(PN 9816)
Union Scientific Corp.
9633 Liberty Road
Randallstown, MD 21133
From Applied Biosystems:
• MicroAmp™ Splash
Free 96-Well Base
(PN 4312063)
Tel: (410) 496-0200
Fax: (410) 521-4590
[email protected]
www.unionscientific.com
10
BigDye® XTerminator™ Purification Kit Protocol
Materials
Table 5
Vortexers and adapters for 384-well plates
Vortexer and Source
Digital Vortex-Genie 2
See Table 4 on page 9 for recommended
models and source.
Adapter or Accessories
If you have another model
of Digital Vortex-Genie 2,
you need:
• Shock absorbing feet
(PN 0K-0400-900)
• Microplate adapter for
Applied Biosystems
(includes 2 elastic
bands) (PN SI-0513)
• Replacement elastic
bands (2) for Microplate
adapter for Applied
Biosystems (PN 0K0513-900)
If you use the Recessed
Platform
(PN 504-0039-00):
• Elastic bands (PN 5680001-00)
IKA MS 3 Digital (PN 3319000)
This vortexer includes the MS 3.4
Microtiter Attachment (PN 3426400).
See Table 4 on page 9 for source.
From any office supply
vendor:
• Elastic bands, size 64
(90 mm ! 6 mm)
Note: For regions outside
North America, an electrical
plug adapter may be
required.
Union Scientific Vertical Shaker
(PN 9816)
None
See Table 4 on page 9 for source.
Taitec MicroMixer E-36 (PN BNE36)
None
See Table 4 on page 9 for source.
BigDye® XTerminator™ Purification Kit Protocol
11
Materials
Table 5
Vortexers and adapters for 384-well plates (continued)
Vortexer and Source
MixMate, 120 V, 50/60 Hz
(PN 022674200)
Adapter or Accessories
None
MixMate, 240 V, 50/60 Hz
(PN 022674226)
Eppendorf USA
One Cantiague Road
P.O. Box 1019
Westbury, NY 11590-0207
Tel: (800)-645-3050
[email protected]
www.eppendorf.com
Other Materials
and Accessories
Table 6
Other materials and accessories
Item
Source
Swinging-bucket centrifuge
Major laboratory supplier
Troughs (if using multichannel pipettors)
Major laboratory supplier
ALPS 300™ Automated Laboratory Plate
Sealer (optional)
ABgene Inc., USA
565 Blossom Road
Rochester, NY 14610
Note: You can also use Applied
Biosystems MicroAmp ™ Clear Adhesive
Films to seal your plates.
Tel: (585) 654-4800
Fax: (585) 654-4810
[email protected]
abgene.com
12
BigDye® XTerminator™ Purification Kit Protocol
Materials
Table 6
Other materials and accessories (continued)
Item
Source
Wide-orifice (>1.0 mm) pipette tips such
as:
• Rainin Wide-Orifice LTS 250-µL Tip
960/10 (PN RT L250W)
Rainin Instrument, LLC
7500 Edgewater Drive
P.O. Box 2160
Oakland, CA 94621-0060
Tel: (510) 564-1600
Fax: (510) 564-1617
Rainin Instrument, LLC
Rainin Road, Box 4026
Woburn, MA 01888-4026
Tel: (781) 935-3050
Fax: (510) 564-1617
[email protected]
www.rainin.com
• Axygen wide-bore tips
(PN T-205-WB-C-R)
Axygen Scientific Inc.
33210 Central Ave.
Union City, CA 94587
Tel: (510) 494-8900
Fax: (510) 494-0700
[email protected]
www.axygen.com
BigDye® XTerminator™ Purification Kit Protocol
13
Before You Begin Purification
Before You Begin Purification
The BigDye® XTerminator™ Purification Kit consists of two
reagents: XTerminator Solution and SAM™ Solution. The insoluble
phase of the reagent mixture separates if left undisturbed during
sample preparation. It is important to deliver a homogeneous mixture
of the reagents to each well so that correct proportions of the reagents
are present.
The pipetting method (sequential or premix) affects the steps
required to keep the two phases of the BigDye XTerminator reagents
adequately mixed. Protocols for sequential, premix, and robotic
pipetting follow.
Software
IMPORTANT! If you are doing direct injection, verify that the
appropriate software run module for your system is installed and
updated (see Appendix A, “Run Modules,” on page 37). You can
download the run modules from www.appliedbiosystems.com.
The BigDye XTerminator run modules adjust the injection height to
removed only the supernatant containing the purified dye labeled
extension products to inject in the sequencer. In addition, the
injection times and voltages are optimized for the higher signal
usually seen with samples purified with the BigDye XTerminator
Purification Kit.
Equipment and Materials
• Obtain the required materials (see Table 2 on page 6)
• Select a vortexer (see Tables 4 and 5, starting on page 9).
– If you use the Digital Vortex-Genie 2, read “Mixing with the
Scientific Industries Digital Vortex-Genie 2” on page 47.
– If you use the Union Scientific Vertical Shaker, read “Mixing
with the Union Scientific Vertical Shaker” on page 50.
– If you use the IKA MS3 Digital, read “Mixing with the IKA
MS 3 Digital Vortexer” on page 52.
– If you use the Taitec MicroMixer, read “Mixing with the
Taitec MicroMixer” on page 55.
• Select an instrument and software (see Table 2 on page 6)
• Identify the procedure to follow. If you use:
14
BigDye® XTerminator™ Purification Kit Protocol
Performing Purification – Sequential Pipetting
– Sequential pipetting: Follow the procedure in “Performing
Purification – Sequential Pipetting,” below.
– Premix pipetting: Follow the procedure in “Performing
Purification – Premix Pipetting” on page 20.
– Robotic pipetting: Follow the procedure in Appendix E,
“Performing Purification – Robotic Pipetting.”
Performing Purification – Sequential Pipetting
Guidelines for
Sequential
Pipetting
These guidelines apply to single- and multi-dispense pipettes.
• Use wide-bore pipette tips (tips with an orifice >1.0 mm)
whenever pipetting the XTerminator Solution.
• Use conventional pipette tips for pipetting the SAM Solution.
• Agitate the XTerminator Solution for at least 10 seconds using a
standard laboratory vortexer at maximum speed before
pipetting.
IMPORTANT! XTerminator Solution allowed to stand for more
than 2 minutes must be reagitated.
• When pipetting the XTerminator Solution, place the tip of the
pipettor below the surface of the liquid.
Note: Applied Biosystems recommends that you aspirate no
more than the volume of XTerminator Solution that you can
pipette in approximately 2 minutes.
• Dispense at the fastest speed setting and touch off during
dispensing.
BigDye® XTerminator™ Purification Kit Protocol
15
Performing Purification – Sequential Pipetting
Performing
Purification
This procedure gives instructions for purification using sequential
addition of the BigDye XTerminator Purification Kit reagents. The
SAM Solution is added first, followed by the XTerminator Solution.
CHEMICAL HAZARD. SAM Solution is a
flammable liquid and vapor. It may be harmful if absorbed through
the skin, inhaled or swallowed. Exposure may cause eye, skin, and
respiratory tract irritation, liver damage, and central nervous system
depression. Read the MSDS, and follow the handling instructions.
Wear appropriate protective eyewear, clothing, and gloves.
1. After cycle sequencing is complete, centrifuge the reaction
plate for 1 minute.
2. To each well of the reaction plate, add the volume of SAM
Solution specified below using a conventional pipette tip.
Make sure there are no particulates in the SAM Solution
before pipetting. If particulates are present, heat the SAM
Solution to 37 "C and mix to resuspend. Cool to room
temperature before using.
Plate Type and Reaction
Volume/Well
Volume of SAM™
Solution/Well (µL)
384-well, 5-µL
22.5
96-well, 10-µL
45
96-well, 20-µL
90
IMPORTANT! For 384-well reactions with reaction volume
less than 5 µL, add water to bring the volume to 5 µL before
adding SAM Solution. For 96-well reactions with reaction
volume less than 10 µL, add water to bring the volume to
10 µL before adding SAM Solution.
16
BigDye® XTerminator™ Purification Kit Protocol
Performing Purification – Sequential Pipetting
3. Add the XTerminator Solution:
a. Vortex the XTerminator Solution bulk container at
maximum speed for at least 10 seconds, until it is
homogeneous.
b. Use a wide-bore pipette tip to aspirate the XTerminator
Solution.
IMPORTANT! Avoid pipetting from the top of the
liquid.
c. Into each well, add the volume of XTerminator Solution
specified below:
Plate Type and Reaction
Volume/Well
Volume of XTerminator™
Solution/Well (µL)
384-well, 5-µL
5
96-well, 10-µL
10
96-well, 20-µL
20
d. Discard the pipette tip.
4. Seal the plate using:
• A heat seal at 160 "C for 1.5 seconds (see Table 6 on
page 12 and the note below).
or
• MicroAmp™ Clear Adhesive Films (follow the
procedure in Appendix C).
Verify that each well is sealed.
IMPORTANT! If you use a 3730/3730xl instrument and plan
to use direct injection without a septa mat, only Applied
Biosystems Heat Seal Film for Sequencing and Fragment
Analysis Sample Plates (PN 4337570) is supported.
5. Place the plate in a recommended vortexer (see Tables 4 and
5, starting on page 9). Use the appropriate adapter, if
recommended.
BigDye® XTerminator™ Purification Kit Protocol
17
Performing Purification – Sequential Pipetting
6. Vortex the reaction plate for 30 minutes, using the following
conditions:
Vortexer
Plate Type
Digital Vortex-Genie 2
Speed
96-well
1800 rpm
384-well
2000 rpm
Eppendorf MixMate
384-well
2600 rpm
IKA MS3 Digital
Either
2000 rpm ‡
IKA Vortex 3
Either
Setting 5 §
Taitec MicroMixer E-36
Either
Maximum
Union Scientific Vertical
Shaker #
Either
Setting 100
‡ Set the vortexer to Mode B. See “Mixing with the IKA MS 3
Digital Vortexer” on page 52 for instructions.
§ Use the maximum setting that does not cause the vortexer to
“walk” across the bench.
# Add any additional plates to meet mass requirements. (See
“Mixing with the Union Scientific Vertical Shaker” on page 50 for
information.)
It is recommended to pause vortexing after 1 minute and
examine the wells to verify that the contents are well mixed.
7. In a swinging-bucket centrifuge, spin the plate at 1000 ! g
for 2 minutes.
At this point you can analyze the samples or store the sealed
plates for later analysis (see “Storage of Purified Samples”
on page 27).
18
BigDye® XTerminator™ Purification Kit Protocol
Performing Purification – Sequential Pipetting
8. Place the reaction plate in the instrument.
IMPORTANT! Do not heat-denature or use Hi-Di™
Formamide with samples containing BigDye XTerminator
reagents.
• For 384-well plates and a:
– 3730/3730xl instrument – If the plate is heat sealed,
place it directly in the instrument. Otherwise, remove
the adhesive film, transfer 10 µL of the supernatant
to a clean plate, cover the plate with a septa mat, then
place it in the instrument.
– 3100/3100-Avant, 3130/3130xl or 310 Genetic
Analyzer instrument – Remove the adhesive film or
heat seal, transfer 10 µL of the supernatant to a clean
plate, cover the plate with a septa mat, then place it in
the instrument.
• For 96-well plates and a:
– 3730/3730xl instrument – If the plate is heat sealed,
place it directly in the instrument. Otherwise, remove
the adhesive film, cover the plate with a septa mat,
then place it in the instrument.
– 3100/3100-Avant or 3130/3130xl instrument, with
Data Collection software v2.0 or later– Remove
the adhesive film or heat seal, cover the plate with a
septa mat, then place it in the instrument.
– 3100/3100-Avant instrument with Data Collection
software v1.x or 310 Genetic Analyzer – Remove
the adhesive film or heat seal, transfer 10 µL of the
supernatant to a clean plate, cover the plate with a
septa mat, then place it in the instrument.
9. In the Data Collection Software Plate Editor, select the
appropriate run module:
• For most plate and instrument combinations – Select
the BigDye XTerminator run module for your
instrument and plate type (see Appendix A, “Run
Modules”).
• If you transferred the supernatant to a clean plate
after vortexing – Select a standard run module.
10. Run the plate.
BigDye® XTerminator™ Purification Kit Protocol
19
Performing Purification – Premix Pipetting
Performing Purification – Premix Pipetting
If you use premix pipetting:
• Prepare a mixture of the two BigDye® XTerminator™ reagents
(referred to here as “premix”).
• Perform the purification, according to the procedure on page 23.
For purification using robotic pipetting, see Appendix E,
“Performing Purification – Robotic Pipetting.”
Guidelines for
Preparing the
Premix
These guidelines apply to single- and multi-dispense pipettes.
• Use wide-bore pipette tips (tips with an orifice >1.0 mm) for
pipetting the XTerminator Solution.
• Use conventional pipette tips for pipetting the SAM Solution.
• Agitate the XTerminator Solution for at least 10 seconds using a
standard laboratory vortexer at maximum speed before
pipetting.
IMPORTANT! XTerminator Solution that is allowed to stand for
more than 2 minutes must be revortexed.
20
BigDye® XTerminator™ Purification Kit Protocol
Performing Purification – Premix Pipetting
Preparing the
Premix
CHEMICAL HAZARD. SAM Solution is a
flammable liquid and vapor. It may be harmful if absorbed through
the skin, inhaled or swallowed. Exposure may cause eye, skin, and
respiratory tract irritation, liver damage, and central nervous system
depression. Read the MSDS, and follow the handling instructions.
Wear appropriate protective eyewear, clothing, and gloves.
1. Based on your plate and reaction size, calculate the volumes
of XTerminator Solution and SAM Solution needed.
Volume/Well (µL)
Plate Type and
Reaction Volume/Well
XTerminator™
Solution
SAM™ Solution
384-well, 5 µL
5
22.5
96-well, 10 µL
10
45
96-well, 20 µL
20
90
Multiply the number of reactions to be performed by the
volume of XTerminator Solution and SAM Solution needed
per reaction, adding 10 to 20% extra volume for reagent
trough dead volume. The ratio of SAM Solution to
XTerminator Solution should be 4.5 to 1 (v/v).
For example, for one 96-well, 10-µL reaction plate with 10%
extra volume:
• XTerminator Solution:
10 µL/reaction ! 96 reactions ! 1.10 = 1056 µL
• SAM Solution:
45 µL/reaction ! 96 reactions ! 1.10 = 4752 µL
BigDye® XTerminator™ Purification Kit Protocol
21
Performing Purification – Premix Pipetting
2. Combine the SAM Solution and the XTerminator Solution:
a. Vortex the XTerminator Solution bulk container at
maximum speed for at least 10 seconds, until it is
homogeneous.
b. Using a wide-bore pipette tip or a graduated cylinder,
transfer the appropriate volume of XTerminator
Solution to a clean container.
IMPORTANT! Insert the pipette tip well below the
surface of the liquid before aspirating.
c. Using a conventional pipette tip or graduated cylinder,
add the appropriate volume of SAM Solution to the
container with the XTerminator Solution.
Make sure there are no particulates in the SAM
Solution before pipetting. If particulates are present,
heat the SAM Solution to 37 "C and mix to redissolve.
Cool to room temperature before using.
d. Mix the reagents until homogenous.
3. Depending on your schedule:
• Continue to “Performing Purification” on page 23.
or
• Store the premix in a clean, capped container at 4 "C for
up to 5 days.
Guidelines for
Pipetting the
Premix
• Use conventional pipette tips when pipetting the premix.
• When pipetting from a bottle, keep the premix agitated using a
rocking motion.
IMPORTANT! Using a stir bar on a stir plate does not keep the
premix properly suspended.
• When pipetting from a trough, keep the premix agitated by:
– Rocking the trough back and forth lengthwise to create a
wave motion.
or
– Placing the pipette tips 1 to 2 mm above the trough bottom
and moving them gently from side-to-side.
• Agitate the XTerminator premix before each aspiration.
22
BigDye® XTerminator™ Purification Kit Protocol
Performing Purification – Premix Pipetting
Performing
Purification
This procedure provides instructions for purification by single-step
addition of the premix and automated pipettors.
1. Be sure the premix is well mixed, then transfer it to the
trough or reservoir.
2. After cycle sequencing is complete, centrifuge the reaction
plate for 1 minute.
BigDye® XTerminator™ Purification Kit Protocol
23
Performing Purification – Premix Pipetting
3. Into each well of the reaction plate, use a conventional
pipette tip to add the volume of premix specified below:
Plate Type and Reaction
Volume/Well
Volume of Premix/Well
(µL)
384-well, 5-µL
27.5
96-well, 10-µL
55
96-well, 20-µL
110
Add more premix to the trough or reservoir as necessary.
IMPORTANT! For 384-well reactions with reaction volume
less than 5 µL, add water to bring the volume to 5 µL before
adding SAM Solution. For 96-well reactions with reaction
volume less than 10 µL, add water to bring the volume to
10 µL before adding SAM Solution.
IMPORTANT! Dispense the premix within 1 minute of
aspiration to avoid separation of the reagents in the pipette
tip.
4. Seal the plate using:
• A heat seal at 160 "C for 1.5 seconds (see Table 6 on
page 12 and the note below).
or
• MicroAmp™ Clear Adhesive Films (follow the
procedure in Appendix C).
Verify that each well is sealed.
IMPORTANT! If you use a 3730/3730xl instrument and plan
to use direct injection without a septa mat, only Applied
Biosystems Heat Seal Film for Sequencing and Fragment
Analysis Sample Plates (PN 4337570) is supported.
5. Place the plate in a recommended vortexer (see Tables 4 and
5, starting on page 8). Use the appropriate adapter, if
recommended.
24
BigDye® XTerminator™ Purification Kit Protocol
Performing Purification – Premix Pipetting
6. Vortex the reaction plate for 30 minutes using the following
conditions:
Vortexer
Digital Vortex-Genie 2
Plate Type
Speed
96-well
1800 rpm
384-well
2000 rpm
Eppendorf MixMate
384-well
2600 rpm
IKA MS3 Digital
Either
2000 rpm ‡
IKA Vortex 3
Either
Setting 5 §
Taitec MicroMixer E-36
Either
Maximum
Union Scientific Vertical
Shaker #
Either
Setting 100
‡ Set the vortexer to Mode B. See “Mixing with the IKA MS 3
Digital Vortexer” on page 52 for instructions.
§ Use the maximum setting that does not cause the vortexer to
“walk” across the bench.
# Add any additional plates to meet mass requirements. (See
“Mixing with the Union Scientific Vertical Shaker” on page 50 for
information.)
It is recommended to pause vortexing after 1 minute and
examine the wells to verify that the contents are well mixed.
7. In a swinging-bucket centrifuge, spin the plate at 1000 ! g
for 2 minutes.
At this point you can analyze the samples or store the sealed
plates for later analysis (see “Storage of Purified Samples”
on page 27).
BigDye® XTerminator™ Purification Kit Protocol
25
Performing Purification – Premix Pipetting
8. Place the reaction plate in the instrument.
IMPORTANT! Do not heat-denature or use Hi-Di™
Formamide with samples containing BigDye XTerminator
reagents.
• For 384-well plates and a:
– 3730/3730xl instrument – If the plate is heat sealed,
place it directly in the instrument. Otherwise, remove
the adhesive film, transfer 10 µL of the supernatant
to a clean plate, cover the plate with a septa mat, then
place it in the instrument.
– 3100/3100-Avant, 3130/3130xl or 310 Genetic
Analyzer instrument – Remove the adhesive film or
heat seal, transfer 10 µL of the supernatant to a clean
plate, cover the plate with a septa mat, then place it in
the instrument.
• For 96-well plates and a:
– 3730/3730xl instrument – If the plate is heat sealed,
place it directly in the instrument. Otherwise, remove
the adhesive film, cover the plate with a septa mat,
then place it in the instrument.
– 3100/3100-Avant or 3130/3130xl instrument, with
Data Collection software v2.0 or later– Remove
the adhesive film or heat seal, cover the plate with a
septa mat, then place it in the instrument.
– 3100/3100-Avant instrument with Data Collection
software v1.x or 310 Genetic Analyzer – Remove
the adhesive film or heat seal, transfer 10 µL of the
supernatant to a clean plate, cover the plate with a
septa mat, then place it in the instrument.
9. In the Data Collection Software Plate Editor, select the
appropriate run module:
• For most plate and instrument combinations – Select
the BigDye XTerminator run module for your
instrument and plate type (see Appendix A, “Run
Modules”).
• If you transferred the supernatant to a clean plate
after vortexing – Select a standard run module.
10. Run the plate.
26
BigDye® XTerminator™ Purification Kit Protocol
Storage of Purified Samples
Storage of Purified Samples
Samples purified with the BigDye® XTerminator™ Purification Kit
can be stored under the following conditions:
• Room temperature storage – Sample plates sealed with heat
seal film, adhesive film, or septa for up to 48 hours at room
temperature (20 to 25 "C).
• Refrigerated storage – Sample plates sealed with heat seal film
or adhesive film for up to 10 days at 4 "C.
• Frozen storage – Sample plates sealed with heat seal film or
adhesive film for up to 10 days at #20 "C.
BigDye® XTerminator™ Purification Kit Protocol
27
Troubleshooting
Troubleshooting
Observation
Possible Cause
Recommended Action
Dye artifacts
(blobs) at
beginning of
electropherogram
Incomplete mixing during purification
due to:
• Non-recommended vortexer used
Use recommended mixing equipment.
• Vortexer speed too low
• Mixing time insufficient
• Mixing not uniform across plate
• Follow mixing guidelines in
Appendix D.
• Stop vortexer after 1 minute and
examine wells to verify that mixing is
complete.
Insufficient SAM Solution
Use 4.5 parts SAM™ Solution for every
1 part XTerminator™ Solution.
SAM Solution cloudy
See “SAM Solution forms precipitates”
on page 34.
Leaking plate seal
• If using clear adhesive film, follow
the sealing protocol in Appendix C.
• If using a heat seal, increase the
time and temperature for the
heat-sealing step.
Insufficient DNA template in reaction
Increase DNA template concentration
(see Appendix B).
Insufficient BigDye® XTerminator™
reagent due to:
28
• Incorrect volume added
Use a volume of XTerminator Solution
equal to the volume of the reaction to
be purified.
• BigDye XTerminator reagent not
properly suspended during
dispensing
• Pipette tip with incorrect bore size
used
Follow guidelines for mixing and
dispensing XTerminator Solution on
page 15.
• XTerminator Solution pipetted
from the very top of the container
When pipetting XTerminator Solution,
be sure the pipette tip is below the
surface of the liquid.
BigDye® XTerminator™ Purification Kit Protocol
Troubleshooting
Observation
Offscale signal
Weak signal
Possible Cause
Recommended Action
Excessive DNA injected due to:
• Extension product concentration
too high
• Reinject samples as soon as
possible. Second injections from
samples with offscale data
frequently give acceptable onscale
data.
• Decrease DNA template
concentration (see Appendix B).
• Injection time too long
• Injection voltage too high
Decrease injection time and/or voltage.
Insufficient DNA injected due to:
• Extension product concentration
too low
Increase DNA template concentration
(see Appendix B).
• Failed sequencing reaction
Repeat sequencing reaction.
• Injection time too short
• Injection voltage too low
Increase injection time and/or voltage.
Incorrect injection height due to:
• Non-BigDye XTerminator
Purification Kit run module used
Use the correct BigDye XTerminator
Purification Kit run module (see
Appendix A).
• Excessive XTerminator Solution
causing incorrect bed height
Use correct amount of XTerminator
Solution.
• BigDye XTerminator Purification
Kit run module offset not updated
after autosampler recalibration
Run the Update BDx Utility to update
run module offset values (see
Appendix A).
• Non-supported plate
Use supported plates.
BigDye® XTerminator™ Purification Kit Protocol
29
Troubleshooting
Observation
Weak signal
Possible Cause
Recommended Action
Incomplete desalting due to:
• Insufficient XTerminator Solution
used
Use a volume of XTerminator Solution
equal to the volume of the reaction to
be purified.
• Incomplete mixing
• Follow guidelines for mixing and
dispensing XTerminator Solution on
page 15.
• Follow mixing guidelines in
Appendix D.
• When pipetting XTerminator
Solution, be sure the pipette tip is
below the surface of the liquid.
Insufficient volume in well due to:
• Insufficient SAM Solution used
during purification
Use 4.5 parts SAM Solution for every 1
part XTerminator Solution.
• Pipetting with wide-bore pipette
tips created by manually cutting
conventional pipette tips
Purchase and use wide-bore (orifice
>1.0 mm) pipette tips.
• Leakage of seal during purification
• If using clear adhesive film, follow
the sealing protocol in Appendix C.
• If using a heat seal, increase the
time and temperature for the
heat-sealing step.
Capillary electrophoresis system
issues, such as:
Troubleshoot capillary electrophoresis
system.
•
•
•
•
30
Old array
Expired polymer
Old buffer
Wrong mobility file
XTerminator Solution overheated
Do not expose BigDye XTerminator
reagents to temperatures above 25 "C.
Hi-Di™ Formamide used or sample
heat denatured
Follow the protocol and do not use
Hi-Di™ Formamide or heat denature the
sample.
BigDye® XTerminator™ Purification Kit Protocol
Troubleshooting
Observation
Delayed analysis
start point
(Slow migrating
samples/late
starts)
Poor
resolution/short
read lengths
Possible Cause
Recommended Action
Unlabeled DNA template injected
into capillary, interfering with proper
separation of extension products due
to:
• Too much template in sequencing
reaction
• High-molecular-weight template
used in sequencing reaction
• Reinject samples as soon as
possible. Second injections from
samples with offscale data
frequently give acceptable onscale
data.
• Decrease template concentration in
DNA sequencing reaction (see
Appendix B).
• Injection time too long
• Injection voltage too high
Decrease injection time and/or voltage.
Offscale signal
See “Offscale signal” on page 29.
Degradation of purified extension
products due to:
• Samples stored for >48 hours at
room temperature
• Samples stored for >10 days in
refrigerator or freezer
Prepare fresh sample and analyze
within 48 hours or store appropriately.
• Stored samples improperly sealed
Seal samples with adhesive or heat
seal film for storage.
• Insufficient SAM Solution used
during purification
Use 4.5 parts SAM Solution for every 1
part XTerminator Solution.
• Old or improperly sealed septa
used
Use fresh septa mat when running
plates on DNA sequencer.
Insufficient signal strength
See “Weak signal” on page 29.
Capillary electrophoresis system
issues, such as:
Troubleshoot capillary electrophoresis
system.
•
•
•
•
Old array
Expired polymer
Old buffer
Wrong mobility file
Injection of high-molecular-weight
template DNA
BigDye® XTerminator™ Purification Kit Protocol
See “Delayed analysis start point (Slow
migrating samples/late starts)”, above.
31
Troubleshooting
Observation
Loss of small
extension
products
Possible Cause
Recommended Action
Over-exposure of purified extension
products to BigDye XTerminator
reagent due to:
• Mixing during purification in great
excess of recommended time
Follow the protocol.
• Sample stored for >48 hours at
room temperature
• Sample stored for >10 days in
refrigerator or freezer.
Prepare fresh sample and analyze
within 48 hours, or store appropriately.
Degradation of BigDye XTerminator
reagents due to:
32
• Exposure of reagents to high
temperature (>30 "C), even briefly,
before or during purification
• Exposure of reagents to room
temperature for more than 24
hours
Repurify samples and ensure that
samples remain at room temperature
during processing.
• Reagent shelf-life exceeded
Discard and use fresh BigDye
XTerminator reagents.
Protocol not optimized for small
molecule recovery.
• Use POP-6™ polymer for capillary
electrophoresis.
• Use the BigDye® Terminator v.1.1
Cycle Sequencing Kit.
• Load reaction plates as soon as
possible after centrifuging.
BigDye® XTerminator™ Purification Kit Protocol
Troubleshooting
Observation
No signal
Possible Cause
Recommended Action
Incorrect injection height due to:
• Non-BigDye XTerminator
Purification Kit run module used
Use the correct BigDye XTerminator
Purification Kit run module (see
Appendix A).
• Excessive XTerminator Solution,
causing incorrect bed height
Use correct amount of XTerminator
Solution.
• BigDye XTerminator Purification
Kit run module offset not updated
after autosampler recalibration
Run the Update BDx Utility to update
run module offset values (see
Appendix A).
• Nonsupported plate
Use supported plates.
Insufficient volume in well due to:
• Insufficient SAM Solution used
during purification
Use 4.5 parts SAM Solution for every 1
part XTerminator Solution.
• Leakage of seal during purification
• If using clear adhesive film, follow
the sealing protocol in Appendix C.
• If using a heat seal, increase the
time and temperature for the
heat-sealing step.
Degraded BigDye XTerminator
reagent due to extreme exposure to
heat
See “Loss of small extension products”
on page 32.
Extremely delayed analysis start
point; slow migrating samples
See “Delayed analysis start point (Slow
migrating samples/late starts)” on
page 31.
Failed sequencing reaction
Repeat the sequencing reaction.
Capillary electrophoresis system
issues, such as:
Troubleshoot the capillary
electrophoresis system.
•
•
•
•
Old array
Expired polymer
Old buffer
Wrong mobility file
Hi-Di™ Formamide used, or sample
heat denatured
BigDye® XTerminator™ Purification Kit Protocol
Follow the protocol and do not use
Hi-Di™ Formamide or heat denature the
sample.
33
Troubleshooting
Observation
SAM™ Solution
cloudy
Possible Cause
Recommended Action
SAM Solution forms precipitates
• Gently warm SAM Solution to 25 to
37 "C and mix to redissolve. Allow
to cool to room temperature before
using. Warm SAM Solution
produces poor results.
Note: If SAM Solution is mixed
vigorously, allow foam to settle
before using.
• Store SAM Solution at room
temperature.
Leaking wells
Plate poorly sealed during
purification.
• If using clear adhesive film, follow
the sealing protocol in Appendix C.
• If using a heat seal, increase the
time and temperature for the
heat-sealing step.
Nonuniform
purification results
across plate
Nonuniform mixing across plate due
to:
• Non-recommended vortexer used
Use recommended mixing equipment.
• Vortexer speed too low
• Follow mixing guidelines in
Appendix D.
• Stop vortexer after 1 minute and
examine wells to verify that mixing is
complete.
Nonuniform dispensing of BigDye
XTerminator reagent across plate due
to:
Follow guidelines for mixing and
dispensing XTerminator Solution (see
page 15) or premix (see pages 20 and
22).
• XTerminator Solution not
completely suspended during
dispensing
• Pipette tip with incorrect bore size
used
• Premix not properly suspended
during dispensing
XTerminator
Solution and/or
SAM™ Solution
frozen
34
Reagents stored improperly.
Gently thaw reagents in refrigerator.
Allow reagents to thaw completely
before mixing.
BigDye® XTerminator™ Purification Kit Protocol
Troubleshooting
Observation
Possible Cause
XTerminator
Solution too thick
to pipette
Improper pipetting technique due to:
• XTerminator Solution not
completely suspended during
dispensing
• Pipette tip with incorrect bore size
used
Evaporation
BigDye® XTerminator™ Purification Kit Protocol
Recommended Action
Follow guidelines for mixing and
dispensing XTerminator Solution on
page 15.
• Discard and use fresh XTerminator
Solution.
• Seal the XTerminator Solution
storage bottle properly.
• Implement evaporation control for
extended dispensing.
35
Troubleshooting
36
BigDye® XTerminator™ Purification Kit Protocol
Appendix A: Run Modules
Overview
This appendix provides:
• A list of BigDye® XTerminator™ Purification Kit run modules
• Instructions for installing, updating, and uninstalling BigDye
XTerminator Purification Kit run modules
The BigDye XTerminator run modules adjust the injection height to
removed only the supernatant containing the purified dye labeled
extension products to inject in the sequencer.
When analyzing purified samples, select the run module appropriate
to your system components from Table 1.
Note: If you transfer the supernatant to a clean plate after
centrifuging, refer to the instrument User Guide for the appropriate
run module.
Table 1
BigDye® XTerminator™ Purification Kit run modules
Instrument
3730/3730xl
DNA Analyzer
Array length
(cm)
Polymer
KB QV20 LOR
50
POP-7™
900
BDx_XLRSeq50_POP7
850
BDx_LongSeq50_POP7
700
BDx_FastSeq50_POP7
700
BDx_StdSeq36_POP7
550
BDx_RapidSeq36_POP7
36
POP-7™
BigDye® XTerminator™ Purification Kit Protocol
Appropriate Software
Run Module
37
Table 1
BigDye® XTerminator™ Purification Kit run modules
Instrument
Array length
(cm)
Polymer
KB QV20 LOR
80
POP-7™
950
BDx_LongSeq80_POP7
POP-4™
700
BDx_LongSeq80_POP4
POP-7™
850
BDx_StdSeq50_POP7
POP-6™
600
BDx_StdSeq50_POP6
POP-4™
600
BDx_StdSeq50_POP4
POP-7™
700
BDx_FastSeq50_POP7
POP-7™
600
BDx_RapidSeq36_POP7
POP-6™
500
BDx_RapidSeq36_POP6
POP-7™
500
BDx_UltraSeq36_POP7
POP-4™
400
BDx_UltraSeq36_POP4
ABI PRISM®
3100/3100Avant,
3130/3130xl
Genetic
Analyzer
50
36
Installing Run
Modules
Appropriate Software
Run Module
Applied Biosystems provides BigDye XTerminator Purification Kit
run modules for the 3100/3100-Avant, 3130/3130xl, and 3730/3730xl
Data Collection Software. Use these run modules whenever you run a
plate containing BigDye XTerminator reagents. The modules adjust
the sample injection height to prevent injection failures.
IMPORTANT! If you pipette the purified supernatant from a reaction
plate to a new reaction plate, use a standard run module instead of a
BigDye XTerminator run module to inject from the new plate.
1. Download the Support Files Installer:
a. Go to www.appliedbiosystems.com
b. Click Support, then Software Downloads.
c. In the list, select BigDye® XTerminator™ Purification
Kit.
d. Click the
link next to the BigDye XTerminator
Support Files Installer to download the installer file.
2. Power on the instrument (solid green light state) and the data
collection computer.
38
BigDye® XTerminator™ Purification Kit Protocol
3. Quit the data collection software if it is running.
4. Double-click BDx_Installer.exe, then follow the prompts in the
Support Files Installer wizard.
Updating Run
Modules
You need to update the run modules with new calibration values
whenever the sequencer autosampler is recalibrated. You run the
Update BDx Utility to update the BDx run modules.
1. Quit the Data Collection software, but do not shut down the data
collection computer.
2. Power on the instrument (solid green light state).
3. Select Start!Programs!Applied Biosystems!BDx
Utility!Update BDx Utility.
Uninstalling Run
Modules
Note: Uninstalling the BDx run modules also removes any
instrument protocols and plate records that use a BDx run module.
Run folders containing ab1 files that have been extracted to the hard
drive are not removed.
1. Power on the instrument (solid green light state) and the data
collection computer.
2. Quit the data collection software if it is running.
3. Select Start!Programs!Applied Biosystems!BDx
Utility!Uninstall BDx.
4. Follow the prompts in the Uninstaller wizard.
BigDye® XTerminator™ Purification Kit Protocol
39
40
BigDye® XTerminator™ Purification Kit Protocol
Appendix B: DNA Quantity Guidelines
DNA sequencing reactions purified with the BigDye® XTerminator™
Purification Kit result in high signal strength when analyzed on a
DNA sequencer. When you prepare sequencing samples for
purification with the BigDye XTerminator reagents, you may need to
decrease the amount of DNA template in the sequencing reactions to
keep the fluorescence signals on scale during analysis.
When you plan to purify samples with BigDye XTerminator kit, use
the following table as a guide to the amount of template DNA for the
initial cycle sequencing.
• Start at the lower end of the range. If low signal strength is
observed, increase the quantity.
• If offscale signal is observed, decrease the template
concentration.
IMPORTANT! If you decrease the template concentration, also
decrease the amount of any template controls proportionately. For
example, if you run a pGEM control, you should dilute it 1:2 or 1:4
and add only 1 to 2 µL.
Table 1
DNA template quantity guidelines
Template Type
DNA Quantity/Reaction (ng)
PCR products
100 to 200 bp
0.5 to 3
200 to 500 bp
1 to 10
500 to 1000 bp
2 to 20
1000 to 2000 bp
5 to 40
>2000 bp
10 to 50
Other types of template
Single-stranded DNA
BigDye® XTerminator™ Purification Kit Protocol
10 to 50
41
Table 1
DNA template quantity guidelines (continued)
Template Type
Double-stranded DNA
Cosmid or BAC DNA
Bacterial genomic DNA
42
DNA Quantity/Reaction (ng)
50 to 300
200 to 1,000
1,000 to 3,000
BigDye® XTerminator™ Purification Kit Protocol
Appendix C: Plate Sealing Procedure
IMPORTANT! Always handle the MicroAmp™ Clear Adhesive Film
by the end-tabs.
To use the Clear Adhesive Film for the BigDye® XTerminator™
purification protocol:
1. Place the loaded reaction plate in the appropriate Splash Free
Support Base and wipe away any drops of XTerminator™ Solution
or SAM™ Solution that may have spilled on the sealing surface of
the plate.
Note: Any liquid on the surface of the plate before the Clear
Adhesive Film is applied causes inadequate sealing between the
film and plate and may result in leakage.
2. Remove a single Clear Adhesive Film from the bag. Notice the
two ends of the film are without adhesive.
3. Peel back the white protective backing from the center sealing
surface in one swift movement, taking care not to touch the
center sealing surface with your fingers.
4. With the Clear Adhesive Film positioned over the reaction plate,
gently lower the film onto the plate. Handle only the nonadhesive ends of the film.
BigDye® XTerminator™ Purification Kit Protocol
43
5. While applying firm pressure, drag the applicator slowly across
the Clear Adhesive Film, side to side and top to bottom, to
ensure good contact between the film and the plate over the
entire surface of the reaction plate.
Note: Applicators are available separately (PN 4333183).
6. Repeat step 5. With firm pressure, drag the applicator at least 5
times side to side and 5 times top to bottom, with firm pressure,
over the Clear Adhesive Film for a leak-free seal.
7. Using firm pressure, run the edge of the applicator along all four
sides of the outside border of the Clear Adhesive Film following
the lines in the figure below.
44
BigDye® XTerminator™ Purification Kit Protocol
8. Using a paper tissue and firm pressure, run a finger across each
column and row of the plate to ensure each well is tightly sealed.
Optionally, place the plate in a thermal cycler, close the lid and
program the thermal cycler to hold 20 "C for 5 minutes.
Ensure the sample block temperature is less than 25 "C.
The heat and pressure from the thermal cycler lid completes the
sealing process.
Continue with the purification protocol.
BigDye® XTerminator™ Purification Kit Protocol
45
46
BigDye® XTerminator™ Purification Kit Protocol
Appendix D: Mixing Guidelines
Mixing with the Scientific Industries Digital VortexGenie 2
• If needed, replace the stock rubber feet with the shock-absorbing
feet (see Table 4 on page 9). To remove the stock rubber feet,
turn the vortexer upside down and remove the feet using a
1/4-in. hex driver. Use the 1/4-in. hex driver to install the
shock-absorbing feet.
• If needed, attach the Microplate Adapter for Applied
Biosystems (PN SI-0513) to the vortexer. When installing the
adapter, push from the center to avoid bending the adapter.
• Secure the plate to the adapter using the elastic bands as shown
in Figure 1 on page 48.
• Be sure that the plate is well secured to the platform before
starting the vortexer.
• Take care not to disrupt the plate sealing film when attaching the
plate to the platform. If the seal is disturbed, remove the seal and
replace with a fresh seal.
• If you use the Digital Vortex-Genie 2 with the recessed platform
adapter (PN SI-0513), the position of the elastic band varies,
depending on the type of plate you use:
– For 96-well plates, see Figure 2 on page 48.
– For 384-well plates, see Figure 3 on page 49.
BigDye® XTerminator™ Purification Kit Protocol
47
Mixing with the Scientific Industries Digital Vortex-Genie 2
Figure 1 Elastic band positions for either type of plate with the
Microplate Adapter for Applied Biosystems
Figure 2 Elastic band position for 96-well plates with the
recessed platform adapter
48
BigDye® XTerminator™ Purification Kit Protocol
Mixing with the Scientific Industries Digital Vortex-Genie 2
Figure 3 Elastic band position for 384-well plates with the
recessed platform adapter
BigDye® XTerminator™ Purification Kit Protocol
49
Mixing with the Union Scientific Vertical Shaker
Mixing with the Union Scientific Vertical Shaker
Note: The Union Scientific vertical shaker can be noisy when it
shakes. If the shaker is placed on the floor, it operates more quietly.
Loading
Additional Plates
The Union Scientific vertical shaker is designed to operate optimally
with a total sample weight (plates, samples, and bases) between 450
and 2500 g.
If the mass of your filled plates is less than 450 grams, add additional
plates to the vertical shaker until the total mass is greater than 450
grams.
Use Table 1 to calculate the mass of your filled plates and any
additional masses needed for the vertical shaker. See the example
calculation after Table 1.
Table 1
Masses of reaction plates and reagents
Item
Mass (g)
MicroAmp ™ Optical 384-Well Reaction Plates‡
Empty
18
Reagent mass per well
0.033
Reagent mass per plate
12
Total mass of filled plate, including reagents
30
MicroAmp ™ Optical 96-Well Reaction Plates
Empty
22
MicroAmp™ Splash Free 96-Well Base
68
10-µL reactions
Reagent mass/well
0.065
Reagent mass/plate
6
Total mass of filled plate
20-µL reactions
50
Reagent mass/well
28
0.130
Reagent mass/plate
12
Total mass of filled plate
34
BigDye® XTerminator™ Purification Kit Protocol
Mixing with the Union Scientific Vertical Shaker
‡ 384-well plates are used without bases. 96-well plates require bases.
Example
Calculation
Suppose you have three 96-well plates with 10-µL reactions.
The total mass of the plates, the reagents, and the splash-free bases is
288 grams [(3 ! 68) + (3 ! 28) = 288 grams].
162 more grams are required to make a total mass of 450 grams.
(450 # 288 = 162 grams).
Any combination of plates and bases that total to at least 162 grams is
sufficient, such as:
• Three empty bases [(3 ! 68) = 204 grams]
or
• Two plates, each with a base [(2 ! 22) + (2 ! 68) = 180 grams]
BigDye® XTerminator™ Purification Kit Protocol
51
Mixing with the IKA MS 3 Digital Vortexer
Mixing with the IKA MS 3 Digital Vortexer
IMPORTANT! The vortexer must be set to Mode B before use. See
“Set the Vortexer Mode” on page 54.
• In regions outside North America, an electrical plug adapter
may be required.
• The IKA Microtiter Attachment (PN MS 3.4) is supplied with
the IKA MS 3 vortexer and is required for 96- or 384-well plates
(see Figure 4).
Follow the instructions in the MS 3 Users Manual to install the
microtiter adapter.
• Follow the loading instructions appropriate for your plate:
– For 96-well plates, secure the front and rear of the 96-well
plate with elastic bands, as shown in Figure 5.
– For 384-well plates, flex the plate slightly lengthwise and slip
the front and rear of the plate under the tabs (see Figure 6).
Figure 4
52
IKA MS3 Microtiter Attachment
BigDye® XTerminator™ Purification Kit Protocol
Mixing with the IKA MS 3 Digital Vortexer
Figure 5 IKA MS 3 with a 96-well plate, showing placement of
the elastic bands
Figure 6 Close-up of the IKA MS3 with a 384-well plate, showing
a tab holding the plate
BigDye® XTerminator™ Purification Kit Protocol
53
Mixing with the IKA MS 3 Digital Vortexer
Set the Vortexer
Mode
The MS 3 has two speed ranges: Mode A (low-speed) and Mode B
(high-speed). For the BigDye® XTerminator™ Purification Kit, you
need to set the vortexer to Mode B.
Each time the vortexer powers on, it is in Mode A. To set the mode to
Mode B each time you use the vortexer, hold down the Start/Stop
button, then press the Power button (see Figure 7).
This changes the vortexer to Mode B, with a top speed of 3000 rpm.
Power button
!
1
Start
Stop
Start/Stop button
Figure 7
54
IKA MS 3 Digital front panel
BigDye® XTerminator™ Purification Kit Protocol
Mixing with the Taitec MicroMixer
Mixing with the Taitec MicroMixer
1. Load the plates
• For 96-well plates, put the plate into a MicroAmp™
96-Well Base (PN N8010531), then use the white levers to
clamp the plate onto the MicroMixer.
• For 384-well plates, use the white levers to clamp the plate
directly onto the MicroMixer.
2. Set the Range switch to High Speed.
3. Turn the Speed dial all the way to the right, for maximum speed.
Clamp
Range switch
Figure 8
Speed dial
Taitec MicroMixer control panel
BigDye® XTerminator™ Purification Kit Protocol
55
Mixing with the Taitec MicroMixer
56
BigDye® XTerminator™ Purification Kit Protocol
Appendix E: Performing Purification –
Robotic Pipetting
Overview
This appendix provides materials and instructions for purification
with the BigDye® XTerminator™ Purification Kit using robotic
pipettors.
If you use robotic pipetting:
1. Prepare a mixture of the two BigDye® XTerminator™ reagents
(referred to here as “premix”).
2. Perform the purification, according to the procedure below.
Materials
Table 1
I
Robotic pipetting systems and accessories
Item
Biomek FX/NX robotic pipetting
system, with either:
• 96-mandrel, 200-µL head
• 384-mandrel, 30-µL head
Source
Beckman Coulter
4300 N. Harbor Boulevard
P.O. Box 3100
Fullerton, CA 92834-3100
Tel: (800) 742-2345
Fax: (800) 643-4366
www.beckmancoulter.com
Bubble Paddle Stirrer for Biomek
FX/NX (PN VP OK21Kit)
V&P Scientific, Inc.
9823 Pacific Heights Boulevard
Suite T
San Diego, CA 92121
Tel: (858) 455-0643
Fax: (858) 455-0703
[email protected]
www.vp-scientific.com
BigDye® XTerminator™ Purification Kit Protocol
57
Table 1
Robotic pipetting systems and accessories (continued)
Item
Source
Pipette tips
From Beckman Coulter:
Beckman Coulter (see above)
• For the 96-mandrel head, 96-well
tips (PN 717252)
• For the 384-µL mandrel head:
– 384-well 30-µL tips
(PN 719223)
– 384-well 50-µL tips
(PN A22288)
From Axygen:
• For the 96-mandrel head, 96-well
tips (PN FX-250-L-R-S)
• For the 384-mandrel head:
– 384-well 30-µL tips
(PN FX-384-L-R-S)
– 384-well 50-µL tips
(PN FX-384-XL-L-R-S)
58
Axygen Scientific Inc.
33210 Central Ave.
Union City, CA 94587
Tel: (510) 494-8900
Fax: (510) 494-0700
[email protected]
www.axygen.com
BigDye® XTerminator™ Purification Kit Protocol
Performing
Purification
1. Prepare the appropriate amount of premix, as described in
“Preparing the Premix” on page 21.
2. Program the robotic pipettor with the mode, speed, and air
gap:
a. Set the mode:
Pipetting Head
Channels
Wells/Plate
Mode
96
96
Single aspiration/
single dispense
96
384
Single aspiration/
multi-dispense
384
384
Single aspiration/
single dispense
b. Set the aspiration speed to 30 µL/sec and the dispense
speed to 90 µL/sec.
c. Set the pipettor to preaspirate at least 5 µL of air (for
blowout after dispensing).
BigDye® XTerminator™ Purification Kit Protocol
59
3. Continue programming the robotic pipettor:
a. Set the position of the tips for aspiration, based on the
stirring mode your pipettor supports:
– If stirring can be stopped during aspiration, position
the tips at least 2 mm above the trough bottom.
– If stirring cannot be stopped during aspiration,
position the tips at least 10 mm above the stirring
mechanism.
Note: Damage to the pipetting head can occur if the
tips contact the stirring mechanism. Verify that
sufficient clearance is maintained between the pipetting
head and the stirring mechanism.
b. Set the blowout volume.
Note: If you use a 96-well pipetting head to dispense
into 384-well plates, you can use multi-dispensing to
dispense into all quadrants from a single aspiration,
followed by a blowout step after the last dispense.
c. Set the dispense height no more than 2 mm lower than
the top of the reaction plate well and enable tip touch.
4. Mix and then transfer the premix to the reservoir.
5. Run the method to aspirate and dispense the premix.
Add premix to the reservoir as needed.
6. Seal the plate using:
• A heat seal at 160 "C for 1.5 seconds (see Table 6 on
page 12 and the note below).
or
• MicroAmp™ Clear Adhesive Films (follow the
procedure in Appendix C).
Verify that each well is sealed.
IMPORTANT! If you use a 3730/3730xl instrument and plan
to use direct injection without a septa mat, only Applied
Biosystems Heat Seal Film for Sequencing and Fragment
Analysis Sample Plates (PN 4337570) is supported.
60
BigDye® XTerminator™ Purification Kit Protocol
7. Place the plate in a recommended vortexer (see Tables 4 and
5, starting on page 9). Use the appropriate adapter, if
recommended.
8. Vortex the reaction plate for 30 minutes, using the following
conditions:
Vortexer
Plate Type
Speed
96-well
1800 rpm
384-well
2000 rpm
Eppendorf MixMate
384-well
2600 rpm
IKA MS3 Digital
Either
2000 rpm ‡
IKA Vortex 3
Either
Setting 5 §
Taitec MicroMixer E-36
Either
Maximum
Union Scientific Vertical
Shaker #
Either
Setting 100
Digital Vortex-Genie 2
‡ Set the vortexer to Mode B. See “Mixing with the IKA MS 3
Digital Vortexer” on page 52 for instructions.
§ Use the maximum setting that does not cause the vortexer to
“walk” across the bench.
# Add any additional plates to meet mass requirements. (See
“Mixing with the Union Scientific Vertical Shaker” on page 50 for
information.)
Note: It is recommended to pause vortexing after 1 minute
and examine the wells to verify that the contents are well
mixed.
9. In a swinging-bucket centrifuge, spin the plate at 1000 ! g
for 2 minutes.
At this point you can analyze the samples or store the sealed
plates for later analysis (see “Storage of Purified Samples”
on page 27).
BigDye® XTerminator™ Purification Kit Protocol
61
10. Place the reaction plate in the instrument.
IMPORTANT! Do not heat-denature or use Hi-Di™
Formamide with samples containing BigDye XTerminator
reagents.
• For 384-well plates and a:
– 3730/3730xl instrument – If the plate is heat sealed,
place it directly in the instrument. Otherwise, remove
the adhesive film, transfer 10 µL of the supernatant
to a clean plate, cover the plate with a septa mat, then
place it in the instrument.
– 3100/3100-Avant, 3130/3130xl or 310 Genetic
Analyzer instrument – Remove the adhesive film or
heat seal, transfer 10 µL of the supernatant to a clean
plate, cover the plate with a septa mat, then place it
in the instrument.
• For 96-well plates and a:
– 3730/3730xl instrument – If the plate is heat sealed,
place it directly in the instrument. Otherwise, remove
the adhesive film, cover the plate with a septa mat,
then place it in the instrument.
– 3100/3100-Avant or 3130/3130xl instrument, with
Data Collection software v2.0 or later– Remove
the adhesive film or heat seal, cover the plate with a
septa mat, then place it in the instrument.
– 3100/3100-Avant instrument with Data Collection
software v1.x or 310 Genetic Analyzer – Remove
the adhesive film or heat seal, transfer 10 µL of the
supernatant to a clean plate, cover the plate with a
septa mat, then place it in the instrument.
11. In the Data Collection Software Plate Editor, select the
appropriate run module:
• For most plate and instrument combinations – Select
the BigDye XTerminator run module for your
instrument and plate type (see Appendix A, “Run
Modules”).
• If you transferred the supernatant to a clean plate
after vortexing – Select a standard run module.
12. Run the plate.
62
BigDye® XTerminator™ Purification Kit Protocol
Worldwide Sales and Support
Applied Biosystems vast distribution and
service network, composed of highly trained
support and applications personnel,
reaches 150 countries on six continents.
For sales office locations and technical support,
please call our local office or refer to our
Web site at www.appliedbiosystems.com.
Applera is committed to providing the
world’s leading technology and information
for life scientists. Applera Corporation
consists of the Applied Biosystems and
Celera businesses.
Headquarters
850 Lincoln Centre Drive
Foster City, CA 94404 USA
Phone: +1 650.638.5800
Toll Free (In North America): +1 800.345.5224
Fax: +1 650.638.5884
07/2007
www.appliedbiosystems.com
Part Number 4374408 Rev. C
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