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.
My baseline is too high (too low). How can I fix this?
Clean the capillary window on the capillary using a lint-free tissue (or use Kimwipe®: tissue) to remove all particulates. After cleaning, perform the Test CCD 4-Color test. This is a 5-minute module and should give you 4 lines in the scan window while the module is running. You should expect to see the lines in the following ranges on a scale of 2,000 on the y-axis: Red and Yellow – 700–1,600 and the Green and Blue – 300–1,000 range.
If your results are not close to these ranges, clean the capillary window again. If that fails, then clean the syringe with warm distilled water and change the polymer using a fresh, unexpired bottle. Replace the polymer in the capillary by using the Seq Fill Capillary module and discard the waste water and replace with fresh water.
If all of the above fails, the problem may be hardware related. Please contact Thermo Fisher Scientific Support to initiate a service call.
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.
