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查看更多产品信息 NED™ Matrix Standard - FAQs (402996)
13 个常见问题解答
You can make a matrix from running the Sequencing Standard or by running Matrix Standards. To make the Matrix:
(1) After the run, open the DNA Sequencing Analysis Software.
(2) Go to the File menu and select Add Sample(s). Select the file(s) of the standards that you ran, click on the “Add Selected Samples” button, then click the OK box.
(3) After the samples have been added to the Sample Manager, click the Show box, and check the raw data. Peak height should be greater than 200 rfu and the baseline needs to be stable.
(4) Go to the “Tools” menu and select “Make Matrix”. Select the number of files you will be generating the matrix from (e.g., if you are generating it from a sample file, select 1; if from Matrix Standards, select 4).
(5) Click on the “…” box to the right of each line to browse to the sample file(s). Select the file and click “Open”. Do this for all of the samples you will use for the Matrix.
(6) In the “Specify the path for the new matrix file” area, name your matrix. The location the file should be stored in should be the default location and in D:\AppliedBio\Shared\Analysis\Basecaller\Matrix.
Note:The matrix file needs to be in both locations in order to autoanalyze the data. If you attempt to point the preferences to look for the matrix in D:\AppliedBiosystems\SeqA5.X\AppSeqA\bin\Basecaller\Matrix, the entire pathway will be written to the .ab1 file for the matrix name and autoanalysis will fail.
(7) Click the “Make Matrix” button. The matrix will either be made correctly or it will generate an error message with specific information on why it failed.
(8) Either copy and paste the Matrix file from the default location to D:\AppliedBio\Shared\Analysis\Basecaller\Matrix or make the matrix 2, the second being saved to that location.
Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Instruments Support Center.
A sequencing matrix can be made by either using a Matrix Standard or a Sequencing Standard. The matrix standards consist of 4 tubes, each representing a different nucleotide/dye that have to be run in order to generate a matrix file for analyzing the data.
To make up the Matrix Standard:
(1) Vortex each of the tubes of Matrix Standard for 1 min at full speed.
(2) Add 1 µL of each Matrix Standard into 12 µL of Hi-Di Formamide (so you should end up with 4 tubes, each containing 13 µL).
(3) Heat the mixture at 95 degrees C for 2 min, then cool rapidly by placing on ice.
(4) Run on the instrument using one of the Sequencing Run modules for Filter Set E*.
The Sequencing Standard is DNA that has been sequenced with the chemistry you want to use (e.g., BigDye Terminator v.3.1), cleaned up, and dried down. After reconstitution, you can run it like a regular sample, use the sample file to make the matrix, and then apply the matrix back to the sample file to see how well it works. The Sequencing Standard can also be used as a positive control to run on your instrument.
To make up the Sequencing Standard:
(1) Add 100 µL of Hi-Di Formamide to a tube containing the lyophilized Sequencing Standard.
(2) Vortex at full speed for at least 1 min (the DNA can be difficult to resuspend, mixing for a shorter time may result in weaker signal and a poor quality matrix).
(3) Heat at 95 degrees C for 2 minutes then cool rapidly by placing on ice.
(4) Run 10 µL using the appropriate mobility file and run module for Filter Set E*.
Note: If making a matrix using the Sequencing Standard, do not use the P4 RapidSeq (1 mL) E module.
(5) If the raw signal intensity is >4000 rfu, take 10 µL of the Sequencing Standard and add it to 90 µL of Hi-Di Formamide and re-run the standard.
*The BigDye Terminator v.1.1 and v.3.1 chemistries are spectrally similar, so they can be run under the same Filter Set. However, you will need a different matrix for each chemistry.
Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Instruments Support Center.
You can make a matrix from either a sample file or by running out matrix standards. If running matrix standards:
After the run, open the ABI PRISM DNA Sequencing Analysis Software. Go to the File menu and select Open Sample from the "Open" menu. Select one of the standards that you ran and check the raw data. Peak height should be greater than 200 rfu and the baseline needs to be stable.
If you are using the ABI PRISM 310 Genetic Analyzer operated by a Windows computer and are using the ABI PRISM DNA Sequencing Analysis Software, the ability to make a matrix has been incorporated into the analysis software. To start, go to the "Sample" menu and select "Make Matrix". Select the number of files you will be generating the matrix from (e.g. if you are generating it from a sample file, select 1; if from matrix standards, select 4) and whether you are doing 4 dye or 5 dye sequencing. Press OK. A window will appear asking you to "Select Sample File (Dye #X) for Matrix Generation".
If you are making a matrix from multiple files (e.g. matrix standards), select the following for ABI PRISM dRhodamine or ABI PRISM BigDye Terminator v.1.1 chemistry:
Dye #1: dR6G
Dye #2: dTAMRA
Dye #3: dROX
Dye #4: dR110
If you are running ABI PRISM BigDye Terminator v.3.1 chemistry, use:
Dye #1: Dye 1
Dye #2: Dye 2
Dye #3: Dye 3
Dye #4: Dye 4
If you are making a matrix from a sample file (e.g. Sequencing Standard), select the sample file. After you have selected the last sample, the program will attempt to generate a matrix file. If it is successful, a window will appear and ask you to type in a name for the matrix and save it. If it is unsuccessful, you will get a message saying it could not generate a matrix. Reasons for this would be either the signal was too weak, too strong, or the wrong sample files were selected for making a matrix.
Once the matrix is made, go to D:\AppliedBiosystems\SeqA5.3.1\AppSeqA\bin\Basecaller\Matrix folder, copy your matrix and paste it in the D:\AppliedBio\Shared\Analysis\Basecaller\Matrix folder so that the matrix will be visible in both the ABI PRISM 310 Genetic Analyzer Data Collection software and in the ABI PRISM DNA Sequencing Analysis Software.
More information on making a matrix can be found on pg. 6-24 to 6-32 in the ABI PRISM 310 Genetic Analyzer User Guide (http://tools.thermofisher.com/content/sfs/manuals/cms_041158.pdf).
Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Software Support Center.
If the peaks in other colors are directly below the peak of interest, the issue could be that the fluorescent dye being used is not part of the selected dye set, the spectral calibration needs to be performed, or the peaks are offscale. Confirm that the dye set selected on the instrument is compatible with the dye being used, run a new spectral calibration if the correct dye set has been selected and, if the signal intensity is too high, decrease sample concentration during PCR or when preparing samples for electrophoresis.
Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.
We do not recommend that you switch dyes in the middle of a project. Switching dyes may result in a shift in the allele sizes as dyes differ in their mobility. If dyes are switched in the middle of a project, it will be necessary to identify the size shift for all the markers/targets in the amplicon product range. The size shift will be consistent throughout the project and it is only necessary to make the adjustment once.
Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.
Both DS-30 and DS-31 matrix standards contain 6-FAM, NED and ROX. The difference is the dye used in the green channel where DS-30 contains the HEX dye versus DS-31 which contains the VIC dye. The VIC dye emits a stronger signal and is more stable than the HEX dye and we suggest that you use the VIC dye for weak amplicons. Both DS-30 and DS-31 are contained in dye set D, as both dyes have excitation and emission wavelengths that are quite similar and, upon the initial release of DS-31 on the Applied Biosystems 310 Genetic Analyzer, it was not necessary to change the filter set to run DS-30 and DS-31
Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.
A dye set is a combination of dyes that have minimum spectral overlap, which will allow you to multiplex products of similar size using different dyes. Although the size ranges may overlap, the use of a different dye will allow the software to easily distinguish between these products.
Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.
We recommend using only Applied Biosystems dyes, as we provide spectral calibration reagents that have been optimized for our dye sets. Non-Applied Biosystems dyes have variable emission spectra and also require a spectral calibration generated for the specific dyes in use to correct for the spectral overlap between the dyes. You are responsible for obtaining the appropriate spectral calibration reagents and for optimizing custom dye sets to ensure that the dye labels do not affect PCR efficiency. The use of dyes outside of the recommended dye sets can result in pull-up and pull-down peaks, which may make allele-calling challenging.
Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.
It is possible to use another company's kit. However, the vendor should be contacted to determine if they have protocol(s) for their kit(s) on the specific capillary electrophoresis (CE) instrument (specifically, the model you intend to use), and if they have reagents to run a spectral calibration or matrix run on the CE instrument with compatibility to the dyes that are part of the kit(s) in question.
Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.
Unfortunately, we do not have fragment analysis-based kits for AFLP analysis. General information regarding AFLP can be found here (https://www.thermofisher.com/us/en/home/life-science/sequencing/fragment-analysis/amplified-fragment-length-polymorphism-aflp-analysis.html).
Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.
We offer the SNaPshot Multiplex Kit for SNP genotyping. Please refer to our overview of SNP Genotyping by Fragment Analysis (https://www.thermofisher.com/us/en/home/life-science/sequencing/dna-sequencing/snp-genotyping-variant-detection-sequencing/snp-genotyping-fragment-analysis.html) for additional information.
Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.
There are several fragment analysis applications that can be run on the CE instruments, such as:
microsatellite analysis, SNP genotyping, fingerprinting, and relative fluorescence quantitation. Please see the DNA Fragment Analysis by Capillary Electrophoresis Guide for more details about these applications (http://tools.thermofisher.com/content/sfs/manuals/4474504.pdf).
Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.
In order to determine the reagents needed, it will be necessary to gather initial information such as the following:
- What is the minimum and maximum amplicon size range?
- Will there be one target per sample or multiple targets (singleplex versus multiplex on the capillary electrophoresis instrument)?
- Is there overlap among the size of amplicons?
This initial information will help identify the spectral calibration needed for the capillary electrophoresis instrument, the fluorescent dye used to label the primer, and the size standard. Additional information can be found in the “Experimental Design” section of the DNA Fragment Analysis by Capillary Electrophoresis Guide (http://tools.thermofisher.com/content/sfs/manuals/4474504.pdf).
Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.