ABI Cleaning and Maintenance, Sample Prep and Running Procedures

**Do NOT use the ABI without being trained by Carmen, Kristin, or Jenny!**

Gel Block and Fittings

  1. In the 310 software: “Window”, then “Manual Control”
  2. Syringe Home à “Execute” – this will raise the syringe pump mechanism, allowing you to unscrew and remove the syringe.
  3. Loosen and remove the three fittings, the buffer reservoir and the capillary from the gel block.
  4. Place the fittings in a beaker of DI water and sonicate for ½ hour.
  5. Gently pull the gel block out of the instrument.
  6. Rinse the channels of gel block with warm tap water by plugging some of the holes. Do this until all traces of polymer are washed from the gel block.
  7. Rinse with DI water and COMPLETELY dry with nitrogen.
  8. Place the gel block back in the instrument by gently pushing it until it pops/clicks/slides into place.
  9. After sonication, COMPLETELY dry the fittings with nitrogen.

Capillary

  1. Cut a new capillary (if necessary) using the smooth side of the capillary cutter.
  2. Use very gentle scoring and then pull apart.
  3. Mark where window is (11 cm from the end) with sharpie.
  4. Put capillary in fitting in gel block, make sure the end of the capillary is in the T cross-section and then tighten the fitting.
  5. Place capillary over window and make sure the sharpie mark lines up with the detection window, then remove the capillary from the instrument:
  6. Place capillary between foil and clamp with tweezers.
  7. Place flame on it until it turns red (capillary should be charred).
  8. Put acetone on a Kimwipe and wipe until all black residue has been removed.
  9. Place capillary back into the instrument and make sure the capillary window aligns with the detection window.
  10. Position the capillary so that the tip slightly below the electrode (~1mm) and use tape to secure the capillary’s position.
  11. In the 310 software, “Instrument”, then “Change Capillary”
  12. Reset the counter to zero.

Buffer, Water and Waste Vials

  1. Prepare 1X ABI buffer:
  2. Aliquot 2 - 700 µL portions of the 10X ABI buffer (located in cold room) into a 15 mL falcon tube.
  3. Add enough DI water to make 14 mL of 1X buffer.
  4. Label glass vials and fill one with 1X ABI buffer and the other with DI water up to the fill line.
  5. Place white and grey caps on the buffer and water vials:
  6. Make sure the cap is dry and the grey is flush with the white.
  7. Push “Tray” button on instrument.
  8. Place ABI buffer in 1 and water in 2.
  9. In 3 goes a 1.5 mL tube with the lid cut off and filled with DI water to approximately the same position as the buffer and water level….this is for waste.
  10. Pour buffer into the buffer reservoir and fill to the red line:
  11. Make sure the buffer is made fresh each time you use it:
  12. You must change buffer once a week.
  13. If things start going bad and you’re using the instrument a lot then change the buffer.
  14. Screw on buffer reservoir (has an O-ring to tighten onto) and make sure you can push down the buffer valve over the reservoir which indicates it’s open.

Filling the Gel Block with Polymer

  1. Remove POP4 polymer from cold room and allow it to reach room temp.
  2. Shake polymer well and let it sit for 5 minutes after shaken:
  3. The syringe is typically filled to around 600 µL.
  4. You’ll need ~100 µL to fill the gel block.
  5. Fill syringe with ~600 µL of polymer and draw up some air.
  6. Clean outside of syringe with water and a Kimwipe.
  7. Tap the syringe gently with your finger or against your palm until any bubbles come loose.
  8. Depress the syringe until there is one drop of polymer on the end of the syringe.
  9. Loosen fitting on capillary and screw in syringe.
  10. Depress syringe until the channels are filled with polymer.
  11. Tighten right fitting first (fitting with capillary).
  12. Tighten bottom fitting next.
  13. Tighten top left fitting last.
  14. If any air bubbles appear in the gel block, then tap in more polymer.
  15. Flip silver switch on syringe pump to the right.
  16. In the 310 software, select “Window” then “Status”:
  17. Black boxes indicate what you’re aiming for and green boxes are what the instrument is at.
  18. Select “Window” then “Manual Control”.
  19. If you tell the instrument to do something in manual control and it doesn’t do it, you need to exit the software and re-open it.
  20. Autosampler to position 3 à “execute”
  21. Autosampler up à “execute” – Until the capillary is submerged in the waste vial.
  22. Buffer valve open à “execute”
  23. Syringe down à “execute” – Start with 100, then 50, then 25, then 10, then 5, and then 1, until the pump has pushed enough polymer through to remove any bubbles.
  24. Buffer valve close à “execute”
  25. Syringe down à “execute” – Make sure it is set to and increment of 1; continue executing until you hear the pump stay on. This will force polymer through the empty capillary.

Autosampler Calibration

  1. In the 310 software, “Instrument”, then “Autosampler Calibration”:
  2. Press “Start”.
  3. Remove autosampler tray and waste tube (in #3).
  4. Press “Resume”.
  5. Position the capillary over the silver dot on the tray:
  6. To lower step size, hold down shift when hitting the key to which way you want to move
  7. Get as close as possible, but not touching.
  8. Press “Set”.
  9. Repeat for dot in back of tray.
  10. Place tray back in and click “Finish”
  11. Close instrument doors.

Running Samples

  1. In the 310 software, “Window”, then “Manual Control”:
  2. Temperature set to 60 à “execute”
  3. To prep samples for injection (you should always include a positive and a negative control, and it is recommended to run an allelic ladder with each set of samples):
  4. Prepare the master mix:
  5. 24.5 µL of HiDi formamide and 0.5 µL of size standard per sample.
  6. Use ROX for CoFiler or ProFiler kits
  7. Use LIZ for IdentiFiler, MiniFiler or YFiler kits
  8. Aliquot 24 µL of master mix into labeled ABI sample tubes.
  9. Pipette 1 µL of sample into the sample tubes.
  10. Place grey cap on, then twist it and push at the same time so the cap is level with the sample tube.
  11. Clean the grey caps by sonicating in DI water for 10 mins.
  12. Pulse vortex and centrifuge all samples.
  13. In the 310 software, “File”, “New”, “GeneScan Sample Sheet 48 tube”
  14. Type in the names of your samples in the order you want them injected.
  15. Select the correct number of dyes for your samples
  16. Use “4 dye” for CoFiler or ProFiler kits
  17. Use “5 dye” for IdentiFiler, MiniFiler or YFiler kits
  18. Save your sample sheet by selecting “File”, “Save As”
  19. Save in the “Sample Sheets” folder
  20. Name the file with your initials and the date.
  21. Press “Tray” on the instrument and load your samples into the appropriate place in the tray. Press “Tray” again to return the tray to its starting position.
  22. Select “File”, “New”, “GeneScan Injection List”
  23. In the drop down menu, look for the sample sheet you just saved.
  24. You shouldn’t have to change any of the run settings, except for the injection time perhaps (no more than 15s for an injection).
  25. Change the matrix to the one that is appropriate for your samples.
  26. 4 dye – select the matrix with the most recent date.
  27. 5 dye – “08ds33”
  28. Click “Run”.
  29. The software will show you the status and test the syringe for leaks.
  30. The current should be between 6-9 μA.
  31. If the current is too high, check buffers and capillary.
  32. Make sure the voltage is at 15 kV.
  33. Background noise should be <1000 RFU.
  34. If it’s not, then the issue is polymer related.
  35. The primer peaks should come out around 2500s.

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