[. . . ] GeneScan Analysis Software
Version 3. 1
User's Manual
© Copyright 1998, The Perkin-Elmer Corporation This product is for research purposes only. GeneScan, Genotyper, and Perkin-Elmer are registered trademarks of The Perkin-Elmer Corporation. ABI, ABI PRISM and the ABI PRISM design, Applied Biosystems, GenoPedigree, PE, and PE Applied Biosystems are trademarks of The Perkin-Elmer Corporation. Sybase is a registered trademark and Sybase SQL Server are trademarks of Sybase, Inc. [. . . ] When you see multiple unexpected peaks of different colors under an expected peak. When you recalibrate your CCD camera (ABI PRISM 310 and ABI PRISM 377) and the change is greater than 3 pixels from the original pixel position. When you replace your filter wheel (ABI 373) or CCD camera (ABI PRISM 310 and ABI PRISM 377).
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Making a Matrix File 6-5
Considerations The following table lists some of the considerations before making a Before Making a matrix file: Matrix File
Consideration How much dye matrix standard to load?Comment With the ABI 373 or ABI PRISM 377, loading more than 3-µL, produces too much signal. Any amount that results in a signal over 4, 000 FU's is too strong. For gel electrophoresis, load the matrix standards with an empty lane between each sample to avoid contamination of the individual dyes by residual material leaking adjacent samples.
After generating a gel image, for ABI 373 and ABI PRISM 377 instruments, check that the tracking of the gel file is adequate.
Where to Store Store matrix files intended for use by Data Collection software in the Matrix Files ABI folder. The ABI folder is located in the System folder on which the
Data Collection software is installed. If Data Collection and Analysis are Performed on Different Computers Make a copy of a matrix and store as follows. This is useful when data collection and analysis are performed on different computers.
Store a copy in the. . . GeneScan Analysis Software.
Note The ABI PRISM® instrument Data Collection software uses the files installed by the GeneScan Analysis Software in the ABI folder. When you run the analysis software, the program also creates several files (such as a Preference file) and an Analysis Log.
6-6 Making a Matrix File
Process of Creating a New Matrix File
Process Diagram The following diagram shows the procedure for making a new matrix
file:
1 Prepare Dye Matrix Standards 2 Load Dye Matrix Standards
9 Evaluate the Matrix File
3 Generate Sample Files
Creating a New Matrix
8 Assign the Matrix File to Select Sample Files 4 View Raw Data
7 Save and Name the Matrix File
5 Choose a Scan Range 6 Generate a New Matrix File
Making a Matrix File 6-7
Steps to Creating a The following table lists the steps to creating a new matrix file: New Matrix
Step 1 2 3 4 5 6 7 8 9 10 11 Process Preparing Matrix Standards for the ABI PRISM 310. See page 6-9 6-12 6-13 6-17 6-20 6-27 6-29 6-32 6-34 6-35 6-37
The Dye Matrix Standard Kits
Table of Kits PE Applied Biosystems provides Dye Matrix Standard kits that are
designed to test and define the multicomponent matrices for specific dye sets. Tubes containing matrix standards for each of the four different dye colors (blue, green, red, and yellow) are available in the each of these kits:
Kit Dye Primer Matrix Standard Kit Fluorescent Amidite Matrix Standards Fluorescent dNTP Matrix Standards Part number 401114 401456 402792
6-8 Making a Matrix File
Preparing Matrix Standards for the ABI PRISM 310
About Formamide !Wear appropriate protective eyewear, clothing, and gloves.
Obtain a copy of the MSDS from the manufacturer. IMPORTANT Use the same matrix standards that you used for your GeneScan Analysis Software run.
The protocol uses formamide as a sample preparation reagent. Fresh formamide must be deionized and aliquotted into smaller volumes for storage. At room temperature, samples in formamide are stable for a maximum of 48 hours. Although not recommended on a routine basis, you can keep samples prepared in formamide frozen for no more than three days, with no detectable loss in resolution, before running on the ABI PRISM® 310 Genetic Analyzer.
Deionizing To deionize formamide: Formamide
Step 1 2 3 4 Action Mix 50-mL of formamide and 5 g of ion-exchange resin (AG501 X8 from BioRad is recommended). Check the pH; it must be 7. 0 9. 0.
Making a Matrix File 6-9
Step 5
Action Make 500-µL aliquots and store them at minus 20°C for up to 3 months.
Preparing the To ensure reproducibility of results for all samples, prepare the Formamide-Size formamide-size standard mix using the 12:1 ratio of reagents stated in Standard Mix the procedure below.
IMPORTANT The formamide-size standard mix for the ABI PRISM 310 differs from the mix prepared for the ABI 373 and ABI PRISM 377, and do not use on other instruments.
!Wash thoroughly after handling formamide.
Prepare the formamide-size standard mix as follows:
Step 1 Action Mix in a sample vial: a. 2 3 4 5 0. 5-µL GeneScan350 [TAMRA] Size Standard (for example). Store the mix at 26 °C until ready to use.
Preparing Matrix About Matrix Standards Standard Samples You must run matrix standards and create a matrix file the first time you
use a new chemistry or change the run conditions. [. . . ] For a brief description of each project option, see "Changing How the Results are Displayed and Printed" on page 7-13.
sample files
Computer files that contain raw and analyzed data. Sample files are created directly by the ABI PRISM 310 and by the GeneScan Analysis Software for the ABI 373 and ABI PRISM 377. Sample files contain data such as peak locations, size calling values, and a record of analysis settings.
scan number (ABI 373 and ABI PRISM 377 instruments) Data Collection software samples data 194 times (194 or 388 times for XL instruments) as it scans across the gel. separation distance Length from the wells of the gel to the read region of the gel. [. . . ]