User Bulletin
ABI P
RISM
®
310 Genetic Analyzer
June 23, 1999 (updated 12/2000)
A Fast Native Protocol for the Analysis of PCR Products
In This Bulletin
This User Bulletin describes the Fast Native Protocol for the separation of DNA
fragments that are
£
500 bp in length.
The following topics are covered in this User Bulletin:
What is the Fast
Native Protocol?
The Fast Native Protocol is a rapid capillary electrophoresis protocol for separating
DNA fragments that are
£
500 bp in length. The Fast Native Protocol is run on the
ABI P
RISM®
310 Genetic Analyzer using a shortened (41-cm) capillary and 7-minute
analysis times.The sizing resolution is at least 5 bp.
Advantages
The advantages of the Fast Native Protocol are as follows:
♦
Separates DNA fragments using 7-minute analysis times
♦
Processes 96 samples in less than 12 hours
♦
Assesses the performance of specific reactions
– PCR, for sizing of fragments that differ by at least 5 bp
– Minisequencing
– Detection of the absence or presence of DNA fragments in a sample
Limitations
The Fast Native Protocol has only been shown to separate DNA fragments that are
£
500 bp in length.
Topic See Page
Safety 2
Before You Begin 3
Material Preparation 4
Preparing for a Run 6
Analyzing the Data 9
Technical Support 12
Page 2 of 16 ABI P
RISM
® 310 Genetic Analyzer
Safety
Documentation User
Attention Words
Five user attention words appear in the text of all Applied Biosystems user
documentation. Each word implies a particular level of observation or action as
follows.
Note
This word is used to call attention to information.
IMPORTANT
This word calls attention to information that is necessary for correct operation of
the kit or instrument.
CAUTION
This word informs the user that damage to the instrument could occur if the
user does not comply with the information. It also indicates a potentially hazardous
situation that could result in minor or moderate injury to the user.
! WARNING !
This word informs the user that serious physical injury to the user or
other persons could occur if these required precautions are not taken.
DANGER
Indicates an imminently hazardous situation that, if not avoided, will result in death
or serious injury
.
General Warning
! WARNING !
CHEMICAL HAZARD. Some chemicals used with Applied Biosystems
instruments or kits may be hazardous and can cause injury, illness, or death. Check
regularly for chemical leaks and chemical spills, which represent potential safety
hazards to the operator. If a leak or a spill occurs, follow clean-up instructions in the
Material Safety Data Sheets (MSDSs), or in the Waste Profile in the Site Preparation and
Safety Guide. Hazardous Chemical Warnings are prominently displayed on the labels of
all hazardous materials. Always read the appropriate MSDSs and Waste Profiles before
using the chemicals in any way.
User Safety
Precautions
Always wear the appropriate protective gloves, clothing, and eyewear when handling
chemicals.
Ordering MSDSs
Material Safety Data Sheets (MSDSs) for hazardous chemicals manufactured by
Applied Biosystems will accompany your first shipment. To receive additional copies of
MSDSs at no extra cost, call Applied Biosystems at (800) 327-3002.
For chemicals required for this instrument but not manufactured or sold by Applied
Biosystems, please obtain the MSDSs from their manufacturers.
Ordering Kits and
Reagents
To order ABI P
RISM®
kits or reagents, please contact Applied Biosystems at one of the
regional sales offices listed in the Technical Support section at the end of this User
Bulletin. Please have the part number of the kit or reagent you are ordering available
when ordering.
ABI P
RISM
® 310 Genetic Analyzer Page 3 of 16
Before You Begin
Materials Required
x
You will need the following materials to perform a Fast Native Protocol run:
Equipment/Reagent Item Source
ABI P
RISM
®
310 Genetic Analyzer
See your local Applied Biosystems
representative
ABI P
RISM
®
310 Data Collection Software, version
1.2 or higher
GeneScan
®
Analysis Software for the 310 Genetic
Analyzer, version 2.0.2 or higher
Glass syringe, 1.0-mL Applied Biosystems
(P/N 4304471)
GeneScan
®
capillary, 47-cm x 50-
m
m i.d. (labeled
with a green mark)
Applied Biosystems
(P/N 401823)
Genetic Analyzer capillary cutters Applied Biosystems
(P/N 401958)
310 GeneScan
®
Polymer (7%) Applied Biosystems
(P/N 401885)
310 Genetic Analyzer Buffer with EDTA Applied Biosystems
(P/N 402824)
Dye Primer Matrix Standards Kit
or
Fluorescent Amidite Matrix Standards Kit
plus NED Matrix Standard
Applied Biosystems
(P/N 401114)
Applied Biosystems
(P/N 401546)
(P/N 402996)
GeneScan
®
-350 Internal Lane Size Standard
or
GeneScan
®
-500 Internal Lane Size Standard
Applied Biosystems
(P/N 401735)
(P/N 401734)
Genetic Analyzer Buffer Vials (4.0-mL) Applied Biosystems
(P/N 401955)
ABI P
RISM
310 Genetic Analyzer User’s Manual
(P/N 903565)
GeneScan Analysis Software User’s Manual
(P/N 904435)
GeneScan Reference Guide
(P/N 4303189)
1.5-mL Eppendorf tube with the lid removed Major Laboratory Suppliers (MLS)
Magnifying glass MLS
Metric ruler MLS
Heat block MLS
Deionized formamide
Note
Use high-quality deionized formamide.
MLS
Page 4 of 16 ABI P
RISM
® 310 Genetic Analyzer
Material Preparation
Shortening the
Capillary
Preparing Stock
Solutions
Prepare stocks of the following reagents/buffers to save time during run setup:
♦
Fast Native Protocol buffer (0.6X 310 Genetic Analyzer Buffer with EDTA)
♦
Fast Native Protocol polymer (2% 310 GeneScan Polymer solution in 0.6X 310
Genetic Analyzer Buffer with EDTA)
Note
Use the Fast Native Protocol buffer and polymer in place of the standard solutions when
running samples.
Making the Fast Native Protocol Buffer
Note
The Fast Native Protocol buffer lasts for 4 weeks at 2–6 °C. Warm the buffer to room
temperature before use.
To cut the capillary:
Step Action
1
Have one of each at hand:
♦
GeneScan capillary (green)
♦
Capillary cutter
♦
Metric ruler
♦
Magnifying glass
2
Align the 41-cm end of the capillary on top of the metric ruler.
3
Using the capillary cutter, score the capillary at 6 cm from the end farthest from the
capillary window.
4
Break off the capillary at the score. The total capillary length is now 41 cm.
Note
Properly dispose of the 6-cm piece of the capillary.
5
Check for a clean cut at the tip with a magnifying glass.
If the cut does not appear clean, smooth the end by lightly brushing the flat end of
the capillary cutter over the end of the capillary.
To prepare 50 mL of Fast Native Protocol buffer:
Step Action
1
To a 50-mL polypropylene tube add:
♦
3.0 mL 310 Genetic Analyzer Buffer with EDTA
♦
47 mL deionized water
2
Mix thoroughly.
ABI P
RISM
® 310 Genetic Analyzer Page 5 of 16
Making the Fast Native Protocol Polymer
Note
Fast Native Protocol polymer lasts for 4 weeks at room temperature.
To prepare 50 mL of Fast Native Protocol polymer:
Step Action
1
To a 50-mL polypropylene tube add:
♦
14.3 mL 310 GeneScan Polymer (7%)
♦
3.0 mL 310 Genetic Analyzer Buffer with EDTA
♦
32.7 mL deionized water
2
Mix thoroughly.
Page 6 of 16 ABI P
RISM
® 310 Genetic Analyzer
Preparing for a Run
Instrument Setup
Refer to the
ABI P
RISM
310 Genetic Analyzer User’s Manual
for the general
procedure.
Setting the Run
Temperature
Set the instrument run temperature to 60 °C immediately before run setup. Refer to
the
ABI P
RISM
310 Genetic Analyzer User’s Manual
for details.
At Any Time Before
a Run
Perform the following at any time before beginning a run:
♦
Complete the Sample Sheet.
Refer to the
ABI P
RISM
GeneScan
Analysis Software Manual
for details.
♦
Create an appropriate Fast Native Protocol matrix file for the chosen dye set.
For directions on preparing matrix samples for Fast Native Protocol analysis, see
“Preparing the Matrix Samples” on page 7.
♦
Create and then reuse a single matrix file for each set of run conditions.
♦
For DNA fragments that are considerably smaller than 500 bp, determine in a trial
run the approximate electrophoresis time in seconds. Then replace the default run
time with this sample-specific run time.
Preparing and
Loading Samples
To prepare and load the samples:
Step Action
1
Label the sample tubes with a permanent marker.
2
To each tube add:
♦
10.5
µ
L deionized formamide
♦
1
µ
L GeneScan Internal Lane Size Standard (GeneScan-350 or GeneScan-500
recommended)
♦
1
µ
L sample
Note
When analyzing DNA fragments that are considerably smaller than 500 bp,
determine in a trial run the approximate electrophoresis time in seconds. Then
replace the default run time with this sample-specific run time.
! WARNING ! CHEMICAL HAZARD. Formamide is a known teratogen
(i.e., it can cause birth defects). Wash thoroughly after handling formamide.
Wear appropriate protective eyewear, clothing, and gloves. Obtain a copy of
the MSDS from the manufacturer.
3Place the samples in a sample tray.
4Place the samples in a heat block or thermal cycler for 5 minutes at 95 °C.
5Remove the samples from the heat source and place immediately in an ice-water
bath.
6Place the sample tray on the autosampler.
Note The 96-well tray used in the GeneAmp® PCR System 9700 requires a tray
adaptor (P/N 4305051) to be used with the ABI PRISM 310 autosampler.
ABI PRISM ® 310 Genetic Analyzer Page 7 of 16
(ll
Preparing the
Matrix Samples
To prepare the matrix samples:
Run Modules The Fast Native Protocol has two dedicated modules: Fast GS Template A and Fast
GS Template D.
Starting the Run
Step Action
1To each of four sample tubes add:
♦11 µL deionized formamide
♦1 µL each of one of the matrix standards
! WARNING ! CHEMICAL HAZARD. Formamide is a known teratogen
(i.e., it can cause birth defects). Wash thoroughly after handling formamide.
Wear appropriate protective eyewear, clothing, and gloves. Obtain a copy of
the MSDS from the manufacturer.
2Place the standards in a heat block or thermal cycler for 5 minutes at 95 ∞C.
3Remove the standards from the heat source and place immediately in an ice-water
bath.
4Run these four injections either alone or in the first four injections of an
experimental run.
The default values for the Fast Native Protocol modules are:
Module Value
Run Time 300 seconds
Electrophoresis Voltage 15 kV
Electrophoresis Temperature 60 °C
Injection Time 5 seconds
Injection Voltage 15 kV
To start the run:
Step Action
1Install a new, 41-cm capillary as described in “Shortening the Capillary” on page 4.
2Select Change Capillary from the Instrument window. Then click OK in the Reset
window to reset the injection counter.
3Set up the Sample Sheet as described in the ABI PRISM 310 Genetic Analyzer
User’s Manual.
Note Prepare the Sample Sheet at any time prior to the run and save it to the
Sample Sheet folder.
4Select New from the File pull-down menu and click the GeneScan Injection List icon.
Page 8 of 16 ABI PRISM ® 310 Genetic Analyzer
5Set up the Injection List as described in the ABI PRISM 310 Genetic Analyzer User’s
Manual.
Be sure to do the following:
♦Select the appropriate Sample Sheet from the Sample Sheet pop-up menu.
♦Select module Fast GS Template A or Fast GS Template D from the Module pop-up
menu for every injection.
IMPORTANT The displayed value in Run Time is in seconds, not minutes. To
change the default value, calculate accordingly.
6Either:
♦Select the appropriate matrix file from the Matrix pop-up menu for every injection.
or
♦Start the run and assign the matrix to the sample files later.
Note If you have not already created a matrix file, see “Preparing the Matrix
Samples” on page 7. Refer to Appendix B of the GeneScan Reference Guide for
instruction on creating a matrix file.
7Click the Start button.
Note If you have not preheated the heat plate, the module has an initial step in
which the plate is heated to the selected run temperature before the first sample is
injected.
To start the run: (continued)
Step Action
ABI PRISM ® 310 Genetic Analyzer Page 9 of 16
Analyzing the Data
Creating a Matrix
File
You must create a matrix file before analyzing the Fast Native Protocol data for the
first time. For more information on creating matrices, refer to Appendix B of the
GeneScan Reference Guide.
For directions on preparing matrix samples for Fast Native Protocol analysis, see
“Preparing the Matrix Samples” on page 7. Be sure to denature the matrix samples
before loading.
To create the matrix file, select the matrix standards and module as shown below.
Note Usually, you create and save a single matrix file for each set of run conditions. However,
if you experience persistent problems, such as spectral overlap in the analyzed data, you
should remake the matrix file even if you have not altered the run conditions.
If using... Use module... And name it...
Dye Primer Matrix
Standards Kit
Fast GS Template A Fast PCR Matrix A
Fluorescent Amidite Matrix
Standards Kit
plus NED Matrix Standard
Fast GS Template D Fast PCR Matrix D
Page 10 of 16 ABI PRISM ® 310 Genetic Analyzer
Setting the Analysis
Parameters
IMPORTANT If you choose to use different analysis parameters from those recommended
here, be sure to define the Analysis Range to exclude the primer peak.
Defining the Size
Standard
Define the size standard so that each unknown peak is flanked by at least two size
standard peaks on either side.
Refer to the GeneScan Analysis Software User’s Manual for detailed protocols.
To set the analysis parameters:
Step Action
1Open the GeneScan Analysis Software (version 2.0.2 or higher).
2Choose New from the File menu and click the Analysis Parameters icon.
3Specify the settings as shown below.
Note When analyzing PCR products, in Analysis Range choose a value for the
start position that begins just after the primer peak.
4Choose Save As... from the File menu.
5Save the settings as Fast Native Analysis Parameters.
6Click OK when done.
ABI PRISM ® 310 Genetic Analyzer Page 11 of 16
Internal Size
Standard
Shown below is the electropherogram for the GeneScan-500 internal lane size
standard, labeled with ROX. Peaks representing 100, 200, 300, 400, and 500 bp are
shaded. The table indicates the electrophoresis time (in minutes) for these peaks.
Note When assigning the size values to the internal size standard peaks, do not assign a value to the 250-bp peak, since
this fragment shows an anomalous migration behavior under the Fast Native Protocol conditions.
Page 12 of 16 ABI PRISM ® 310 Genetic Analyzer
Technical Support
Contacting Technical
Support
You can contact Applied Biosystems for technical support by telephone or fax, by
e-mail, or through the Internet. You can order Applied Biosystems user documents,
MSDSs, certificates of analysis, and other related documents 24 hours a day. In
addition, you can download documents in PDF format from the Applied Biosystems
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To Contact Technical
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Contact technical support by e-mail for help in the following product areas:
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All Other Products 5:30 a.m. to 5:00 p.m. Pacific Time
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Dial...
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then press 8
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then press 21
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then press 22
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GeneScan® applications)
1-800-831-6844,
then press 23
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877 and Catalyst 800 instruments)
1-800-831-6844,
then press 24
1-650-638-5981
ABI PRISM ® 310 Genetic Analyzer Page 13 of 16
Outside North America
ABI PRISM® 3100 Genetic Analyzer 1-800-831-6844,
then press 26
1-650-638-5981
BioInformatics (includes BioLIMS™,
BioMerge™, and SQL GT™
applications)
1-800-831-6844,
then press 25
1-505-982-7690
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1-800-831-6844,
then press 31
1-650-638-5981
Protein Sequencing (Procise‚ Protein
Sequencing Systems)
1-800-831-6844,
then press 32
1-650-638-5981
PCR and Sequence Detection 1-800-762-4001,
then press 1 for PCR,
2 for the 7700 or 5700,
6 for the 6700
or dial 1-800-831-6844,
then press 5
1-240-453-4613
Voyager‰ MALDI-TOF Biospectrometry
and Mariner‰ ESI-TOF Mass
Spectrometry Workstations
1-800-899-5858,
then press 13
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Biochromatography (BioCAD‚
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then press 14
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then press 15
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Peptide Synthesis (Pioneer‰ and 9050
Plus Peptide Synthesizers)
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then press 15
1-508-383-7855
PNA Custom and Synthesis 1-800-899-5858,
then press 15
1-508-383-7855
FMAT‰ 8100 HTS System and
Cytofluor‚ 4000 Fluorescence Plate
Reader
1-800-899-5858,
then press 16
1-508-383-7855
Chemiluminescence (Tropix) 1-800-542-2369 (U.S.
only),
or 1-781-271-0045
1-781-275-8581
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China (Beijing) 86 10 64106608 86 10 64106617
Product or
Product Area
Telephone
Dial...
Fax
Dial...
Page 14 of 16 ABI PRISM ® 310 Genetic Analyzer
Hong Kong 852 2756 6928 852 2756 6968
Korea (Seoul) 82 2 593 6470/6471 82 2 593 6472
Malaysia (Petaling Jaya) 60 3 758 8268 60 3 754 9043
Singapore 65 896 2168 65 896 2147
Taiwan (Taipei Hsien) 886 2 22358 2838 886 2 2358 2839
Thailand (Bangkok) 66 2 719 6405 66 2 319 9788
Europe
Austria (Wien) 43 (0)1 867 35 75 0 43 (0)1 867 35 75 11
Belgium 32 (0)2 712 5555 32 (0)2 712 5516
Czech Republic and Slovakia (Praha) 420 2 61 222 164 420 2 61 222 168
Denmark (Naerum) 45 45 58 60 00 45 45 58 60 01
Finland (Espoo) 358 (0)9 251 24 250 358 (0)9 251 24 243
France (Paris) 33 (0)1 69 59 85 85 33 (0)1 69 59 85 00
Germany (Weiterstadt) 49 (0) 6150 101 0 49 (0) 6150 101 101
Hungary (Budapest) 36 (0)1 270 8398 36 (0)1 270 8288
Italy (Milano) 39 (0)39 83891 39 (0)39 838 9492
Norway (Oslo) 47 23 12 06 05 47 23 12 05 75
Poland, Lithuania, Latvia, and Estonia
(Warszawa)
48 (22) 866 40 10 48 (22) 866 40 20
Portugal (Lisboa) 351 (0)22 605 33 14 351 (0)22 605 33 15
Russia (Moskva) 7 095 935 8888 7 095 564 8787
South East Europe (Zagreb, Croatia) 385 1 34 91 927 385 1 34 91 840
Spain (Tres Cantos) 34 (0)91 806 1210 34 (0)91 806 1206
Sweden (Stockholm) 46 (0)8 619 4400 46 (0)8 619 4401
Switzerland (Rotkreuz) 41 (0)41 799 7777 41 (0)41 790 0676
The Netherlands (Nieuwerkerk a/d
IJssel)
31 (0)180 331400 31 (0)180 331409
United Kingdom (Warrington, Cheshire) 44 (0)1925 825650 44 (0)1925 282502
All other countries not listed
(Warrington, UK)
44 (0)1925 282481 44 (0)1925 282509
Japan
Japan (Hacchobori, Chuo-Ku, Tokyo) 81 3 5566 6006 81 3 5566 6505
Latin America
Del.A. Obregon, Mexico 305-670-4350 305-670-4349
Region
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ABI PRISM ® 310 Genetic Analyzer Page 15 of 16
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© Copyright 2000, Applied Biosystems
For Research Use Only. Not for use in diagnostic procedures.
Applied Biosystems, ABI PRISM, and its design are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other
countries. ABI is a trademark of Applera Corporation or its subsidiaries in the U.S. and certain other countries.
GeneScan and GeneAmp are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries.
All other trademarks are the sole property of their respective owners.
P/N 4312955B, Stock No. 106UB07-02
/
