INSTRUCTIONS FOR AUTOMATED SEQUENCING
JACOBS LAB, UCLA
Before You Begin: Return to menu
1. Check the schedules of other workers in the lab to be sure that the needed equipment will be available.
2. Also check to be sure that the needed solutions and supplies are available.
3. If intending to use the Sequencer, put your name on the calendar in the sequencing lab.
PCR Purification Return to menu
1. Begin heating molecular grade
water in the water bath or a thermal cycler; heat a volume large
enough to provide 50µL per sample to be purified.
2. Start with 43 to 50µL of PCR product.
3. Use an appropriate PCR purification kit & follow the directions
of the kit.
(We currently use Marligen PCR Purification System)
4. At the final step, DNA elution, use 30 - 50µL of warm
(65-70°C) molecular grade water instead of TE buffer.
5. Run 5µL of purified product with 2µL of loading
dye on a gel to check the DNA concentrations of the samples.
Cycle Sequencing Return to menu
1. Set up reactions in 0.2ml tubes.
2. In each tube, add the following:
| 2-5µL of template (depending on the DNA concentration) | |
| 3µL Big Dye (light sensitive so keep in foil!!!) | |
| 1µL Big Dye buffer | |
| 1µL of primer (25 mM) | |
| 0-3µL of molecular grade water to bring total reaction volume to 10µL. |
| Note: Big Dye is light-sensitive; keep the dye covered with foil or in a drawer while preparing samples. After adding Big Dye to samples, also keep the samples out of the light as much as possible. |
3. Place tubes in thermal cycler for:
| 25 cycles of 96C for 10 sec | |
| 50C for 5 sec | |
| 60 for 4 min | |
| followed by 4C indefinitely |
Isopropanol Precipitation Return to menu
1. Transfer the cycle sequencing
product into 1.7mL tubes.
2. Add 80µL of 75% 2-propanol, & let the samples sit
for 20 minutes at room temperature.
3. Spin the samples for 25 minutes at 14000rpm.
4. Remove the supernatant carefully, & discard it.
5. Add 250µL of 75% 2-propanol, & vortex the samples
on low (3-4) for a few seconds.
6. Spin the samples for 5 minutes at 14000rpm.
7. Remove supernatant with a fine pipette tip, being careful not
to disturb or remove the pellet.
8. Speed vac (no heat) or air-dry the samples to remove all alcohol.
Loading Dye & Formamide Return to menu
1. Prepare a cocktail that includes
formamide and loading dye in a 5:1 ratio.
| Note: Prepare enough of the solution to add 3µL to each sample & add "an extra" to account for any pipetting error; e.g., for 10 DNA samples, the total volume of the cocktail would be 33µL ( 27.5µl of formamide & 5.5µL of dye). |
2. Add 3µL of the cocktail to each sample.
Preparing the Sequencing Gel Return to menu
1. Clean the benchtop & adjacent work areas.
2. Place a clean diaper (underpad) on the benchtop where the plates
are to be poured.
| |
3. Use Jacobs Lab's plates. Ensure that plates are clean, free
of dust & marks.
| Note: Use only powder-free gloves. |
4. Place the plate with lateral
notches (at bottom) in the cassette (rig). The outside of the
plate is marked.
5. Spray 2 spacers lightly with dH2O & place 1 on each side,
aligned with the side and bottom of the plate.
6. Get the plate with the medial notch (at top), place it on top,
& clamp the plates to the cassette.
| Note: Be sure the plates are aligned at the top. |
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8. Place plastic tip boxes or equivalent onto the diaper for support
of the glass plates.
9. Get a Gel Pack (Type 373, 48cm WTR) from the cabinet. Ensure the rocker is available.
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10. Follow Gel Pack instructions.
| Note: A single Gel Pack should provide enough material for 2 48-cm gels. |
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| Note: Prevent aerating the mixture when pouring it into the beaker. |
11. Take 35-40ml of polyacrylamide
into the syringe, & tap the syringe to remove air bubbles.
| Note: It works best if you have a mad grin & one eye closed while tapping the syringe. |
13. Insert the back (side opposite teeth) of a plastic comb, &
apply the brace.
14. Wrap the bottom of the plates with a damp paper towel &
Saran wrap to prevent dessication.
Running the Gel & Samples Return to menu
1. Get an ice bucket with ice for cooling the samples after the denaturation step.
2. Turn on the bench-top heating block, & set it to 95°C.
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| Note: Big Dye is light-sensitive; keep the dye covered with foil or in a drawer while preparing samples. After adding Big Dye to samples, also keep the samples out of the light as much as possible. |
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4. Double-click on the ABI-Prism icon.
5. Remove the Saran wrap and paper towels from both ends of the
polymerized gel. Wet the comb with dH2O, & gently remove it.
| Note: Wetting the comb facilitates removal of unwanted pieces of gel. |
7. Create wells by placing a paper comb quickly & evenly into
the space, & add dH2O so that the comb expands.
8. Attach the bottom buffer chamber to the Sequencer, attach its
electrode, and fill it with 1X TBE buffer.
9. Place the cassette (with plates) in the Sequencer, & secure
it with clamps. Close the door.
10. Go to "File" > "New" > "Sequencing
Run". Do a plate check, & then go to "Window"
> "Status" to check the progress of the machine.
| Note: All boxes should be green; if not, there is a problem. Also, if the plates are clean, the results of the plate check should show colored lines near the bottom of the window without any large peaks. If this is not the case, pause the machine, remove the plates and clean them. |
12. Clamp the heating plate onto the outer glass plate, &
plug in its 2 vinyl hoses and electrode.
13. Select "Pre-run". Select "Status" (or
Apple-T) to view progress of machine. The electrophoresis power
should start after 35 seconds, at which point the machine will
produce a whining noise. The heating block will also begin heating
the gel; it will take approximately 35 minutes for the gel to
reach 48 to 50°C.
14. Go to "File" > "New" > "Sequence
Sample," & enter sample information. Save the sample
sheet to your folder (Remember where).
| Note: On the sample sheet, the row number equals the lane of the sequencing gel; i.e., the sample listed in row 12 of the sheet should be loaded into sample well 12 of the gel. Consider leaving some lanes empty at the edges of the gel and between sets of samples so that tracking the lanes on the gel image after sequencing is completed will be easier. |
15. Denature your samples in the heating block at 95°C for
2 minutes, & immediately place them on ice.
16. Flush the lanes of the gel free of bubbles and any debris
using a small syringe.
17. Load 0.8 to 0.9µl of the odd-numbered samples to the
lanes.
| Note: You may use the same pipette tip, if you rinse it well with the buffer between samples. |
18. Close the door, & run the odd-numbered samples for 2 to
5 minutes.
19. Pause the machine, open the door, and load the even-numbered
samples.
20. Close the door, & run the samples into the gel for 10
minutes.
21. Cancel the Pre-run, upload the desired sample sheet, &
ensure that all of the parameters are correct; run time (10 to
12 hours), plate size (48cm), scanning rate (1200/min).
22. Open the door, remove the comb gently, & place the lid
on the top buffer chamber.
23. Close the door, & save the sequencing run (include name
and date).
24. Clean the benchtop, dispose of waste, & put unneeded equipment
away.
Removing the Gel from the Sequencer Return to menu
1. Obtain a large syringe & 500-mL-beaker.
2. Open the Sequencer door, & disconnect all of the electrodes.
3. Remove the lid from the top buffer chamber, & use the syringe
to remove the buffer; use the beaker to collect the buffer.
| Note: Remember this chamber holds the buffer only because it is pressed against the plates; attempting to remove the chamber before emptying the buffer will result in spillage. |
4. Remove the top buffer chamber from the Sequencer, & dump
the buffer down the drain. Re-clamp the top of the plates to the
cassette.
5. Detach the heating plate's hoses from the Sequencer; use KimWipes
to absorb any spillage.
6. Remove the heating plate, & re-clamp the glass plates to
the cassette.
7. Clean the heating plate's surface with dH2O, & place it
face-up in the appropriate drawer.
| Note: Always place the heating plate in the drawer or on the benchtop such that the flat surface clean & faced upward; this surface is that which contacts the sequencing plates. |
8. Remove the cassette (with the
plates) from the Sequencer & place on a diaper (an underpad)
on the benchtop.
9. Remove the bottom buffer chamber; dump the buffer down the
drain.
10. Unclamp the plates from the cassette, lift them out carefully,
& place them on supports (e.g., plastic pipette boxes)
on the benchtop.
11. Remove the spacers, & slide the plates apart.
12. Lay a large KimWipe on the gel, & peel the KimWipe back
to remove the gel from the glass plate. Dispose of the KimWipe
& gel.
13. Wash the plates carefully, using the sink next to the dH2O
carboy; use only dH2O and Alconox.
| Note: Do not use regular dishwashing detergent, as this will leave a film on the Sequencing plates that produces a colorcast and interferes with the sequencing run. Also, do not use paper towels or other abrasive cloths to clean the plates; simply wipe the plates clean by hand with latex gloves. |
14. Clean any gel material &
residue from the Sequencer, but avoid contacting the laser.
15. Clean all other equipment, rinsing all items with dH2O. Wipe
the benchtops clean, & replace diapers (underpads). Be sure
the heating block is turned off.