ABI 310 General Operating Instructions

Tech: Lance Larka
Date: 4-14-98

Chemistries covered:
Dye Terminator
Big Dye Terminator

Comments in red denote vital information.

Contents:

  1. - Choosing your reaction chemistry
  2. - Preparing your samples
  3. - Preparing the ABI 310
    1. - Installing the Capillary
    2. - Changing solutions
  4. - Setting up a run
    1. - Test CCD 4-Color
    2. - Data collection
  5. - Quick Checklist
  1. - Choosing your chemistry

    There are two standard chemistries for sequencing DNA used currently. Dye Terminators and Big Dye Terminators. The principle difference is sequence fidelity and sequence start points. With the DT chemistry, you get sequence anomalies such as "Weak G after A" and good read lengths of 400 bases. With DT chemistry there is a typical loss of the first 10-20 bases after the primer. Keep this in mind when designing your primers. With BDT chemistry, the sequence anomalies are lessened and the length is increased to 450-500 or more. The trade off is that you lose the first 20-40 bases after the primer.

  2. - Preparing your samples

    You must take your sequencing reactions and run them through a G-50 fine sephadex spin column (typically a Centri-sep column) to remove the un-incorporated primers and dye molecules. After running them through the columns you must dry them down in a spin-vac. You must dry the samples down in Perkin Elmer's 0.5ml Sample tubes (P/N 401957). Cap them with PE Septa (P/N 401956). At this point they may be stored at 4 degrees for long periods of time. When you are ready to run your samples on the ABI 310 you must re-suspend the samples with ABI's proprietary "Template Suppression Reagent" (P/N 401674). The allowed range of use is 12-25 ul. I recommend using the lower limit as this improves signal strength and may rescue an otherwise un-analyzable sequence reaction.

    1. Vortex each sample for at least 2 seconds.
    2. Pulse spin. Caution: If you spin too hard the Septa will collapse into the tube.
    3. Heat @ 96 C for 3 minutes.
    4. Vortex each sample briefly.
    5. Pulse spin.
    6. Ready to rock and roll.

  3. - Preparing the ABI

    1. Installing the Capillary

      Every 100 reactions (or so. This will be discussed in the Test CCD section) the Capillary needs to be replaced. Over time the capillary will become 'blocked' with old polymer and fluorescent molecules resulting in poor reads. To change the capillary:

      1. Open Heated block door and laser shield door.
      2. Present Tray
      3. Remove Tape restraining old Capillary
      4. Unscrew the plastic/rubber valve that holds the capillary in the plastic block.
      5. Clean mount thoroughly. Polymer has a tendency to accumulate on the outside and must be removed.
      6. Unscrew capillary holder at tray end.
      7. Save capillary to clean out precipitate accumulations on rubber valve.
        Caution: There is a clear 'window' on the capillary that is susceptible to scratching. Be very careful not to touch it.
      8. Take new capillary out of shipping tube.
      9. Thread the end without the window into the tray side screw-in mount. Line the tip of the Capillary up with the end of the Platinum electrode.
      10. Screw into place tightly.
      11. Thread other end of Capillary through plastic/rubber mount.
        Caution: Unless the capillary is protruding through the end, the mount will be permanently sealed when you screw it into the block. Don't do this.
      12. Screw mount into plastic block very tightly. You should not be able to pull the capillary out of the block. Make sure capillary end is right up against the T-junction between the polymer syringe and the waste vial.
      13. Take Capillary and line the 'window' up with the laser in the small black door. There is a groove that it fits into.
        Caution: The Capillary must be hung perfectly vertical to ensure proper reading.
      14. Tape capillary into place. There should be no abrupt turns or kinks.
      15. Close laser protection window and gel heating door.
      16. Un-present tray.
      17. Close main doors.
      18. Select "Change Capillary" from the "Instrument" pull down menu and click "OK" to reset the use counter.
      19. Ready to Rock and Roll.

    2. Changing the solutions:

      Here is where you have some judgment calls to make. A log on the side of the machine will help here. There are three liquid components to the machine that must be replaced at various times. The Performance Optimized Polymer 6 (P/N 402837), the Genetic Analyzer Buffer (P/N 402824), and the water reservoir (autoclaved DI water). The Buffer and water need to be changed every 2 (recommended) to 3 (for weekends only) days. The polymer needs to be changed every 10 days or after periods of prolonged inactivity. Note: If you have especially finicky sequences you might want to change to polymer before your run.

      1. Changing the Polymer
        1. Remove polymer from refrigerator about an hour before use. There is a precipitate that commonly forms and it must be dissolved before use by letting sit at room temperature. If you are in a hurry you can let it sit in a beaker of room temperature tap water.
        2. Use "Manual Control" to "Syringe Home"
        3. Flush block.
          1. Use Manual control panel under ABI Collection software to "Open Buffer Vial."
          2. Remove Water Syringe.
          3. Remove Capillary screw holder
          4. Remove Polymer Syringe
          5. Pull plastic block straight out.
          6. Remove all fittings and clean with warm water and flush with DI water.
          7. Reinstall all equipment as before.
        4. Squirt polymer from syringe into kimwipe and discard.
          Note: Steps e-h can be omitted if the polymer in the syringe is less than 2 weeks old.
        5. Rinse syringe out with DI water several times.
        6. Draw up about 0.5ml of polymer and then a small air bubble.
        7. invert the syringe several times to let the bubble move from end to end to coat the sides of the syringe. The final inversion should place the bubble at the tip end.
        8. Squirt the bubble, then the polymer into a kimwipe and discard it.
        9. Draw up enough polymer for the number of reactions you wish to run with a maximum of 10 days worth. Draw up an extra 150ul to prime the block. We have determined experimentally that the ABI310 uses 2.5ul of polymer per reaction.
          Caution: Make sure that NO air bubbles are introduced into syringe.
        10. Replace polymer syringe. Screw it in TIGHT. There should be a air bubble in tube in the block at this point. That's good.
        11. Push gently on the polymer syringe plunger until bubble is pushed all the way through the tube.
        12. Use "Manual Control" to close the buffer vial.
        13. Open waste valve fitting. Push polymer through. Close fitting.
        14. Use "Manual Control" to "Syringe Down" until pusher is just barely touching the polymer plunger.

      2. Changing the buffer and water
        1. Dilute 1.5ml of 10X buffer (GAB) with 13.5 ml ddH2O. (Final volume of 15ml)
        2. Rinse out Buffer reservoir from plastic block and 2 buffer vials for tray.
        3. Dry thoroughly.
        4. Fill Buffer reservoir to meniscus line with 1X GAB. Fill Buffer vial to 2mm above meniscus line with 1X GAB. Fill Water buffer vial to 2mm above meniscus line. Replace white plastic caps and put in new septa. Replace all three. GAB vial goes in position 1, H20 vial in position 2.
          Caution: Make very sure that the outside of all liquid containers are bone-dry. Otherwise the machine can arc current through the liquid and damage some very expensive parts.
        5. Take a 1.5ml tube. Cut off lid. Fill to top with ddH20. Place in position 3 in white plastic block.

  4. - Setting up a run

    1. CCD-4 Color check
      1. Do this before every run. This checks to make sure that your capillary isn't getting too old.
      2. Open a new sequence injection list.
      3. Select "Test CCD-4 Color" from the list of sample sheets.
      4. Change "Module" to "Test CCD-4 Color."
      5. "Run"
      6. The levels should all be below 4000 on the "Raw Data" window. If they are higher, try doing a "Sequence Fill Capillary" from the manual control. Then re-do the CCD 4-Color test. If they are still above 4000 replace the capillary.
      7. Close ABI 310 Collection software.
      8. Close Sequencing Analysis software.

    2. Actual Data collection
      1. Start a new session of ABI PRISM 310 Collection.
      2. Create a new Sequence Sample sheet
      3. Enter all data into sheet.
        Important: You must choose the correct "Dye Set/Primer" and "Matrix" selections for the chemistry that you have used. For the Big Dye terminator chemistry, use "DT POP6{BD Set-Any Primer}" and "ch1drhod" respectively. For the Dye Terminator chemistry, use "DT POP6" and POP6 Matrix respectively.
      4. Close the 'unnamed' sheet and save as the default. This will be the current date and time.
      5. Create a new Sequence Injection List
      6. Select the sample sheet that you just made from the list of available sheets.
      7. Set the "Module" to the correct filter set for your chosen chemistry. This will be "Seq POP6(1 mL) E" for the Big Dye chemistry. And "SEQ POP6 A" for the Dye Terminator chemistry. If you do not use the correct settings here, your runs will fail and cannot be recovered.
      8. Open main doors to machine.
      9. Present the tray by pressing the "Tray" button on the left. Be sure to hold the button in until the tray starts to move.
      10. Place the samples in the tray according to their locations listed on the Sample Sheet.
      11. Put the tray back. Close the doors.
      12. Start run.

  5. - Quick checklist

    This section relates to setting up the ABI 310 for use and assumes that your samples are already resuspended in TSR (Section II should already be completed)

    1. If polymer in machine is less than 10 days old and there is enough left for your reactions skip to number 3.
    2. If not, Wash block and fittings, then change polymer. (Section III-B-1).
    3. CCD-4 color check. (See Section IV-A)
      1. If all levels are below 4000 continue on to #4.
      2. If levels are higher than 4000, Do a fresh Gel Pump to fill the capillary with new polymer. Re-run CCD-4 color check. If levels are still above 4000 change the capillary (Section III-A)
    4. Change buffer solution and H20 supply. (Section III-B-2)
    5. Make new Sequence Sample sheet (Section IV-B-1 to 4)
    6. Make new Sequence Injection List (Section IV-B-5 to 7)
    7. Load samples into machine (Section IV-B8 to 11)
    8. Start run. Pray.