310/31xx Genetic Analyzer Sequencing Standards, BigDye™ Terminator v1.1 - FAQs

I see another sequence running under mine. What can cause this?

Some of the causes of another sequence appearing under the primary sequence are:

• Contamination: There is more than one species of DNA present (e.g., multiple PCR products).

• Primers: Primer is annealing in more than one location on the template, primer dimer, primer degradation or not manufactured properly resulting in N+1 or N-1, carry over from PCR reaction, or primers pairs in a multiplex reaction that are not appropriate for multiplexing (i.e., primers anneal inappropriately).

• Spectral/Matrix: If the raw data signal intensity of the sample is too high or saturated it can exceed the amount of color bleedthrough (or spectral overlap) that the matrix (310) or spectral (3130, 3730, 3500) are removing, resulting in secondary peaks appearing in a very specific pattern (e.g., a red peak always appearing under a green peak). A change in the camera, laser, or optical alignment requires that a new matrix be made or a new spectral calibration be performed.

For more information on more than one sequence or set of peaks in a sequencing run, please refer to the DNA Sequencing by Capillary Electrophoresis: Applied Biosystems Chemistry Guide: Second Edition (Cat. No. 4305080, Rev. C). The guide can be found by searching the Thermo Fisher Scientific website with the catalog number 4305080.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

How do I make a sequencing matrix on the 310 Genetic Analyzer?

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.

When trying to make a matrix, I got an error message saying “Not enough peaks to make a matrix”. What happened?

There are two reasons why you might see this message:

Attempting to run Matrix Standards using the P4 RapidSeq (1 mL) E module may result in not having enough peaks being present to generate a successful matrix. If this is the case, try re-running the Matrix Standards using the P4 StdSeq (1 mL) E module or any of the POP-6 modules.

When making the matrix, if you followed the directions for Sequencing Standard but actually ran the Matrix Standards, then you might generate this message since you will have significantly fewer peaks than with Sequencing Standards. Double check the box and tubes that were used and follow the instructions for the standard.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Instruments Support Center.

What do I need to do to get the Sequencing Analysis v.5.X Software to autoanalyze my 310 Genetic Analyzer sequencing samples?

In order to autoanalyze samples on the 310 Genetic Analyzer, there are three things that need to be done:

(1) Copy the Matrix and Mobility files from the Matrix and Mobility folders located at D:\AppliedBiosystems\SeqA5.X\AppSeqA\bin\Basecaller and paste them into the Matrix and Mobility folders at D:\AppliedBio\Shared\Analysis\Basecaller. If you have not made a matrix yet, you will need to do so prior to running samples.
(2) Open Sequencing Analysis, create an Analysis Protocol, and set it as the Analysis Default.
(3) Set up the Preferences to point to the mobility and matrix files in the local directory and in the General Settings tab, point the Autoanalyze with menu to D:\AppliedBiosystems\SeqA5.4\AppSeqA\Automation310.exe

Once the files are copied and the preferences set, you will be ready to run. At the end of the run, the sample files (.ab1) will be analyzed, but you will not see Sequencing Analysis open. The sample files are analyzed with a non-visible version of Sequencing Analysis.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Instruments Support Center.

How do I prepare a matrix standard for sequencing on the ABI PRISM 310 Genetic Analyzer?

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.

How do I make a matrix on the ABI PRISM 310 PC Genetic Analyzer?

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.

What is the shelf life of the sequencing standard?

The sequencing standard has a shelf life of one year from the date of shipment, unless otherwise listed on the CofA. Our Terms and Conditions of sale can be found here(https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/Terms-and-Conditions-of-Sale.pdf).

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Can I re-inject sequencing standard that is already been resuspended in Hi‑Di formamide? If so, how do I store it between uses? How long will it last?

The sequencing standard is intended to be used after resuspension in Hi‑Di Formamide. It can be injected multiple times; the number of times will vary depending on the instrument-specific standard used. This information can be found in the product insert. The sequencing standard should be stable for 24 hours on the instrument.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

How do I make a spectral standard for the Applied Biosystems 3130 series Genetic Analyzer?

The spectral calibration on the Applied Biosystems 3130 series systems can be performed by either using a Matrix Standard or a Sequencing Standard. The Matrix Standards will give 4 peaks, each of a different color whereas the Sequencing Standard is pre-sequenced and cleaned DNA that is dried down and just needs to be resuspended.

To make up the Matrix Standard:

1. Vortex the Matrix Standard tube for 1 min at full speed and pulse spin in a microcentrifuge.
2. Dilute the Matrix Standard in Hi-Di Formamide in a 1.5 mL microcentrifuge tube according to the following dilutions:
   For 4 capillary arrays
   
   • 36 cm and 80 cm arrays: mix 2.5 µL of Matrix Standard with 47.5 µL of Hi-Di Formamide.
   • 50 cm array: mix 2.5 µL of Matrix Standard with 97.5 µL of Hi-Di Formamide.
   
   
   For 16 capillary arrays
   
   • 36 cm and 80 cm arrays: mix 10 µL of Matrix Standard with 190 µL of Hi-Di Formamide.
   • 50 cm array: mix 5 µL of Matrix Standard with 195 µL of Hi-Di Formamide.
3. Mix thoroughly and pulse spin in a microcentrifuge.
4. Denature at 95 degrees C for 2 min and dispense 10 µL of reagent into wells A1–H12 (16 capillaries) or A1–D1 for 4 capillaries on a 96-well plate.
5. Briefly spin to ensure there are no air bubbles at the bottom of the well.
6. Set up and run the spectral calibration as per the Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide