310 Genetic Analyzer with Windows™ OS - FAQs

Polymer in the syringe should be changed every three days. Do not return unused polymer to the bottle.

Buffer and water should be changed every 48 hours or, if the run lasts for more than 48 hours, after the run is complete.

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The electrical requirements for the 310 Genetic Analyzer are:

North America
Voltage – 120 +/- 10% Volts (AC)
Frequency – 50/60 Hz +/- 1%
Circuit Current Protection – 20 A
Power Dissipated (approx). – 1,500 W (5000Btu/hr)

Japan:
Voltage – 100 +/- 10% Volts (AC)
Frequency – 50/60 Hz +/- 1%
Circuit Current Protection – 20 A
Power Dissipated (approx). – 1,500 W (5000Btu/hr)

Europe (pre-1992):
Voltage – 220 +/- 10% Volts (AC)
Frequency – 50/60 Hz +/- 1%
Circuit Current Protection – 10 A
Power Dissipated (approx). – 1,500 W (5000Btu/hr)

Europe (future):
Voltage – 240 +/-10% Volts (AC)
Frequency – 50/60 Hz +/- 1%
Circuit Current Protection – 10 A
Power Dissipated (approx) – 1,500 W (5000Btu/hr)

UK and Australia:
Voltage – 240 + 6%/-10% Volts (AC)
Frequency – 50/60 Hz +/- 1%
Circuit Current Protection – 10 A
Power Dissipated (approx) – 1,500 W (5000Btu/hr)

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The Clear Memory or two-button reset will clear the firmware image, memory, and all calibrated values from the CPU on the 310 Genetic Analyzer. Make sure that all of the calibrated values are known before doing this reset. Usually, these values can be found on a sticker on the inside of the front left door of the instrument.

To perform a Clear Memory reset:

(1) Turn off the instrument and then shut down the computer.

(2) Turn on the instrument while holding both the tray and gel buttons in for 10 seconds. The three lights (Green, Amber, and Red) should all be on, indicating that the firmware image and calibrated values have been erased.

(3) Turn on the computer. The 310 Data Collection should automatically launch and a window should appear that says "Sending Firmware image"(if the 310 Data Collection Software does not automatically launch, manually launch the program). Next, it will ask you to quit the Data Collection application and re-launch the software for this action (resending the Firmware) to take effect.

(4) After opening the 310 Data Collection Software again, open the Manual Control window and input all calibrated values that are listed on the sticker located inside the door (2 - CCD pixel positions, gel pump force, and syringe max travel values). Make sure to click on the "Execute" button after entering values to have them saved on the instrument.

(5) Recalibrate and home the AutoSampler and home the syringe.

Information on how to perform the Clear Memory Reset is also available in Revision B of the 310 Genetic Analyzer User Manual (Cat. No. 4317588), available through the Manuals and Protocols section of the Thermo Fisher Scientific Web site.

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The pump block needs to be cleaned when:

Installing a syringe.

Switching applications (i.e., from sequencing to fragment analysis).

Shutting down the instrument for a long period of time.

If the instrument has run for more than 4 days without any of the above occurring.

Appearance of unusual spikes in the electropherogram.

If the syringe containing the polymer has been on the instrument for more than a week, it is important to clean the syringe and the block, and to change the polymer. At room temperature, enough urea decomposition can occur in one week to cause transient current increases during electrophoreses. If polymer is allowed to dry in the pump block channel, the pump block may be ruined.

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A message will appear at the start of the run telling you if you have enough polymer to complete the run. If you should continue without adding more polymer, then the run will be cancelled at the injection where it ran out and an entry will be made into the run log that there is no polymer. The following injections will appear to run for 1–2 minutes each and will be recorded into the run log. No data will be collected for these injections.

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The Test CCD 4-color module is used to make sure that the capillary window is aligned properly within the detection cell and that the baseline is below 2,000 rfu so that the data is not compromised. Before running the module, make sure that there is polymer in the capillary and that there is sufficient buffer in the buffer vial in position one of the autosampler.

To perform the Test CCD 4-color:

(1) After starting the instrument, open the 310 Data Collection Software.

(2) Prepare a sample sheet and create a new injection list for your run (an old sample sheet can also be used to perform this test).

(3) From within the new injection list, select a sample sheet.

(4) Under Inj.#, click on 1 (first line) and select Insert under the Edit pull-down menu to insert a blank line at the position 1.

(5) Configure the first injection to be the Test CCD 4-color Module

1. Choose any sample name from the pull-down menu in the Inj. Tube & Sample Name column.
2. Choose the Test CCD 4-color module in the Inj #1 Module column.

(6) Start the run by clicking the Run button. The first time you run the test, you will see the 4 colors spike, then drop down and level off. Once they level off, 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
Green and Blue – 300–1,000

If the lines appear higher than that or you only see one line, please see FAQ on “My baseline is too high (too low). How can I fix this?”.

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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.

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.

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Base spacing is roughly defined as the number of scans per base. This value is directly related to the speed at which your samples are traveling through your polymer. The base spacing number will vary depending on the sample quality, capillary length, run module settings, polymer type, how long the polymer was on the system, electrophoresis buffer age, and environment. When you first start running samples, check the spacing to see what the average values are.

If the software is unable to determine the base spacing, it will give a best guess that will appear in red in the base spacing column. Usually this is either due to the data quality or a problem with the sample migration. If you notice the numbers fluctuating, check the following:

-Polymer expiration—if the polymer has expired, wash the block, syringe, and water/waste containers and replace.

-Polymer time on the system—the polymer should be in the syringe on the system for no more than a week. Polymer kept on the system longer than that may compromise the length of read and overall data quality.

-Electrophoresis buffer—the buffer is sold as a 10X concentrate and should be diluted to 1X prior to use. The 1X buffer is good for 1 month.

-Environment conditions—the instrument should be run at ambient temperature. Air flow can also affect migration. Make sure there are no fans, wind from an open window, or air vents blowing directly on the system.

-Sample quality—quantitate the template DNA prior to sequencing. After the sequencing reaction is done, clean the samples using one of the recommended procedures in the BigDye Terminator User Guide. If you are still getting poor results, run the Sequencing Standard as a sample to narrow the focus of your troubleshooting efforts. If the standard looks good, focus on the template and clean up procedures. If the standard looks bad, focus on the system.

If all of the above fail, please contact Thermo Fisher Scientific Technical Support for further assistance.

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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.

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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.

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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.

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The instrument serial number can be found on the back or bottom side of most instruments. For easier customer access, Thermo Fisher Scientific has produced adhesive holders that can be used to display the serial number and history log on the front or side of your instrument. To obtain a holder, please contact your local service engineer, field application specialist, or technical support center.

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You can purchase a computer for installation of the secondary analysis software packages (e.g., Sequencing Analysis, GeneMapper software) as long as the computer meets the software compute requirements (for processor speed, the minimum speed of the processor should be at least 3 GHz) and you can install the software yourself.

For the instrument computer, the computer system has a much more stringent requirement and undergoes thorough testing to make sure it can handle the flow of data from the instrument to the computer. The images for the computer system are streamlined to make sure they have the proper drivers for the computer components, and the Operating System is optimized to make sure there is nothing running in the background that might slow the computer down, take up memory, or conflict with our software. Purchasing a computer commercially can introduce too many variables in terms of hardware and software that can negatively affect the data collection process so we cannot support connecting it to the instrument.

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When placing the capillary on the instrument, if the ferrule is not hand-tightened enough, some polymer gets pushed through the center of the ferrule and/or around the threading. When it dries up, it appears as a white, flaky residue. Use a damp, lint-free cloth to remove it. If more residue appears after the cleanup, it is possible that the ferrule is not tightened down enough or may require replacement.

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If the capillary is stuck in the gel block, even after loosening the ferrule, squirt around the edge of the ferrule with distilled water and try to remove it again. If the capillary will not move, a service call may be required. Do not use excessive force or tools to try and remove it.

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A leak is detected if the gel pump moves a greater distance than expected. Normal polymer usage is approximately 4 - 7 µL per injection which is equivalent to 1 - 2 encoder counts for the 1.0 mL syringe (seen in instrument run log). Actual leaks can occur at various locations on the system and should be carefully examined. The message may sometimes be generated by conditions on the system that mimic a leak - these also need to be considered. More information on troubleshooting this issue can be found on Pages 7 - 9 in the Errors Encountered on the Applied Biosystems 310 Genetic Analyzer Guide (http://tools.thermofisher.com/content/sfs/manuals/cms_042475.pdf).

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Some of the other causes of a fluctuating electrophoresis current error message are:

1.Leak on the system
2. Polymer that:
-Has expired
-Has been left on the instrument for more than the recommended time
-Is a mixture of expired polymer and non-expired polymer
3. Using Running Buffer that was:
-Diluted to 1X incorrectly
-Swapped with the water position on the Autosampler deck
-On the instrument longer than 48 hours
-Not filled to the fill line or evaporated below the fill line
4. An arcing event that was not cleaned afterwards using the water wash wizard
5.Not performing regular maintenance on the instrument
6. Hardware issues

Inspect the system for leaks. If you do not see any leaks on the system, remove the syringe, gel block, anode and cathode buffer containers, water, and rinse containers and clean them with distilled water. Place fresh, non-expired polymer and freshly made 1X Running Buffer on the system. If the problem persists, a service call may be required.

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A perpetually blinking amber light usually indicates that there is a door open on the instrument or a bad door sensor. Open the main door, the oven door and the detector cell door (small black door with the laser warning on it) and close them again. After the autosampler stops moving the light should change to green. On the Applied Biosystems 310 Genetic Analyzer, there is a top panel above the door that can also trigger the blinking amber light. Make sure the panel is pushed all the way in. If the light does not go to green, it is possible that a door sensor is not working properly and a service call may be required.

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A blinking red light on the instrument may indicate that a hardware component has failed or a possible communication disruption has occurred between the instrument and computer. Completely shut down the instrument, wait for 2 minutes, and then start up in the order documented in Applied Biosystems 310 Genetic Analyzer: Starting Up the Instrument Technical Note (https://www.thermofisher.com//content/dam/LifeTech/Documents/PDFs/310%20Start%20Up%20Procedures.pdf) or the Applied Biosystems 310 Genetic Analyzer User Guide (http://tools.thermofisher.com/content/sfs/manuals/cms_041158.pdf).
If the instrument goes from a flashing amber light to red light, one or more of the hardware components has failed the hardware diagnostic and a service call should be opened up.

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If you map the network drive so it shows up under Computer as a drive letter, then this feature can be configured from the Instrument Data Collection Software through the Preferences by going to the Window menu in the Applied Biosystems 310 Genetic Analyzer Data Collection Software Preferences and then changing the “Folder containing Run Folders” location by clicking on the button next to it and browsing to the network folder you will have the files saved in.

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The instrument computer can be connected to your local network via the second NIC card by your local IT group. We don't offer Technical Support to assist with networking issues, and our warranty and service contract terms exclude coverage for instrument system problems that are caused by the network connection (i.e., computer virus, hacking, updates, or patches that alter the compute environment). However, our field service team will respond during the warranty period and while the instrument is covered by an AB Assurance service contract to isolate the cause of any problem either with instrument hardware, software, chemistry, or that arises from an external environmental source including the connection to a customer-owned internal network. Beyond that we can escalate issues to a product technical team who can review instrument logs and advise on the nature of the problem in an effort to help you find and correct the source of the problem. For tips on connecting your computer to the local network, please see the Instrumentation Networking Technical Note (https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/Instrumentation-Networking.pdf).

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All of our systems have been tested with Norton Anti-Virus Software.

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If the instrument is not in use, it is not necessary to replace the buffer every 2 - 3 days. However, the buffer level needs to remain at the fill line to prevent the capillary or array from drying out. Top off the buffer volume with water if necessary. Replace the anode and cathode buffer with freshly made 1X Running Buffer prior to starting a new run.

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For the Applied Biosystems 310 Genetic Analyzer, the buffer can keep for 48 hours or 48 runs.

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Maintenance tasks on the Applied Biosystems 310 Genetic Analyzer take place over the course of 2 - 5 days. For detailed information on the maintenance steps and when to perform them, please refer to the Applied Biosystems 310 Genetic Analyzer Maintenance Checklist Technical Note (https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/310%20Maintenance%20Checklist.pdf) or the Applied Biosystems 310 Genetic Analyzer User Guide (http://tools.thermofisher.com/content/sfs/manuals/cms_041158.pdf).

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The Data Collection and secondary analysis software requires certain permissions in order to function (Read, Write, Delete). To avoid issues with restrictions placed on user accounts, we recommend that you log in either as Administrator or INSTR-ADMIN.

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There is no harm in restarting the system daily, but it should not be necessary unless there is an occasional loss in communication between the instrument and computer or in cases where the system is running significantly slower than normal. If the problems in run speed or communication loss are persistent, then it may be indicative of a larger problem. Technical Support should be contacted for further assistance at techsupport@thermofisher.com.

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We recommend that you perform a restart of the system once a week. The system may require a restart more frequently if you experience issues related to a communication interruption between the instrument and computer. Some systematic issues include:

-The software freezing or becoming unresponsive
-The run timer not counting down or flashing back and forth between two different times
-No data present at the end of the run

In the event of a communication issue between the instrument and computer, close the Data Collection Software (perform a forced shutdown of the application through the Task Manager if Data Collection is unresponsive), completely power down the instrument and turn off the computer. To properly clear the memory, power down the system for at least 2 minutes prior to a restart.

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Perform the following shutdown procedure after doing a short-term or long-term shutdown:

1. Close the Applied Biosystems 310 Data Collection Software and shut down the computer.
2. Turn off the instrument by turning off the switch on the back left-hand side of the instrument. The LED status lights will turn off, and the autosampler will drop.

Short-term shutdown: Store the capillary by keeping one end in the block and the other in the buffer vial (on instrument). NOTE: remove the thermal tape holding the capillary to the heat plate and reposition the capillary into the buffer, then re-attach the thermal tape to secure it.

Long-term shutdown: Uninstall the capillary and properly store it or discard it. If storing the capillary, ensure that both ends of the capillary are submerged in 1x Running Buffer (Genetic Analysis Buffer with EDTA). Maintain the buffer levels in the storage vials so the ends of the capillary are completely submerged.

Step-by-step instructions for the shutdown procedures can be found in the Applied Biosystems 310 Genetic Analyzer: Shutting Down the Instrument Technical Note (https://www.thermofisher.com//content/dam/LifeTech/Documents/PDFs/310%20Shut%20Down%20Procedures.pdf) and the Applied Biosystems 310 Genetic Analyzer User Guide (http://tools.thermofisher.com/content/sfs/manuals/cms_041158.pdf).

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For the Applied Biosystems 310 Genetic Analyzer, if the instrument will be idle for less than a week, perform the short-term shutdown of the system. If it will be idle for longer than a week, follow the instructions in the user guide for a long-term shutdown.

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The Applied Biosystems 310 Genetic Analyzer system includes both the instrument and the computer - you cannot start or restart one without starting the other. Improper boot sequence can lead to communication issues, aborted runs, and possible loss of data. The proper boot sequence is:

1. Remove the thermal tape securing the capillary closest to the electrode and pull the capillary up until the end of the capillary is about 1 mm below the end of the electrode. Failure to do so can cause the capillary to break when the instrument is turned on.
2. Start the computer and the instrument:
- Turn on the computer and log in, but do not launch the Data Collection software.
- Turn on the instrument and wait until you get a solid green light.
- Launch the Data Collection Software.
3. Re-calibrate the autosampler.
4. Home the syringe and refill with polymer if needed.

Step-by-step instructions for the start-up procedures can be found in the Applied Biosystems 310 Genetic Analyzer: Starting Up the Instrument Technical Note (https://www.thermofisher.com//content/dam/LifeTech/Documents/PDFs/310%20Start%20Up%20Procedures.pdf) and the Applied Biosystems 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 Instruments Support Center.