ABI 3730xl Analyzer

**Users Protocol**

> Version Number: 2.3
>
> Production Start Date: 09/10/03
>
> Version 2.3 Date: 7/11/05
>
> Authors: Dan Baker, Chris Daum
>
> Reviewed/Revised by: Jennifer Alvernaz, Cailyn Spurrell

# Summary

This protocol outlines the operation and maintenance of the ABI 3730xl
DNA Analyzer for high throughput capillary electrophoresis.

# Materials & Reagents

+--------------------------------+-------------------+-----------------+
| **[Materials/Rea               | **[Vend           | **[Stock        |
| gents/Equipment]{.underline}** | or]{.underline}** | Number          |
|                                |                   | ]{.underline}** |
| *[Disposables]{.underline}*    | Applied           |                 |
|                                | Biosystems        | 4309849         |
| 384-well Clear Optical         |                   |                 |
| Reaction Plate with Barcode    | Velocity 11       | 06644-001       |
|                                |                   |                 |
| PlateLoc Pierceable Seal       | Corning/VWR       | 430290          |
|                                |                   |                 |
| 50-ml Centrifuge Tube          | Applied           | 4335611         |
|                                | Biosystems        |                 |
| *[Reagents]{.underline}*       |                   | 4335613         |
|                                | Applied           |                 |
| POP-7 Polymer                  | Biosystems        | \-              |
|                                |                   |                 |
| 10X ABI 3730 Buffer with EDTA  | Millipore Milli-Q | \-              |
|                                | System            |                 |
| Milli-Q Water                  |                   | \-              |
|                                | Applied           |                 |
| *[Equipment]{.underline}*      | Biosystems        | \-              |
|                                |                   |                 |
| ABI 3730xl DNA Analyzer        | Velocity 11       | 4335860         |
|                                |                   |                 |
| PlateLoc Thermal Plate Sealer  | Applied           |                 |
|                                | Biosystems        |                 |
| Sealed Array Tool              |                   |                 |
|                                | Applied           |                 |
| Polymer Block Cleaning Kit     | Biosystems        |                 |
+--------------------------------+-------------------+-----------------+

# EH&S

JGI employee performing this procedure must wear a lab coat and gloves.
In situation were there might be a chance of an accidental splash to the
eyes, safety glasses must be worn.

# Workflow

Please refer to the posted daily schedule provided by the ABI Lead
Operator. This schedule will define loading times, reagent swap times,
and walk through and debasing times.

The ABI3730xl is a self-loading high throughput capillary sequencer.
Because of this, instruments are at variable points in their run cycles
at any given time (i.e. there is no batch processing on a system-wide
basis).

ABI3730xl sequencers are concentrated in two locations, room 150 and
room 122. The shift schedules divide tasks between the two locations.

At the end of each shift, operators will send a Daily Update email to
the ABI group at
[**ABI3730xl@cuba.jgi-psf.org**](mailto:ABI3730xl@cuba.jgi-psf.org).
This email will contain room temperatures, a list of errors that
occurred over the course of the shift, and room loading status.

# Procedure

NOTE: All reagents/stock solutions should be prepared prior to the start
of the procedure.

**1. [Launching Data Collection & Sample Sheet Manager
Software]{.underline}**

> ***NOTE:** At the start of the day, all sequencers will be turned on
> and processing plates. This step is NOT required to initiate runs each
> day and is only required following a computer reboot.*

1.  Double-click on the **Run Unified Data Collection 2.0** icon located
    on the desktop. A **Java** window as well as the **Service Console**
    window will be displayed.

> ***NOTE:** Do not close these windows. They are required to run the
> software. You may minimize them. When all the applications are running
> (all green squares in the Service Console) the **Data Collection
> Viewer** window be displayed.*

2.  Click the **+** button to expand the subfolder in the left
    windowpane of the **Data Collection Viewer**. All application
    folders are now visible and ready to access.

3.  Double-click on the **Shortcut to SSManager** icon located on the
    desktop. A Java window (do not close this window) as well as the
    Sample Sheet Manager window will open.

**2. [Pre Run Setup -- Sample Preparation]{.underline}**

> ***NOTE:** Samples should be loaded at the beginning of the day if
> required. See **Workflow** for additional detail.*

1.  Determine how many DNA sample plates to load.

> ***NOTE 1:** A single plate takes approximately 7 hours to run. Refer
> to the loading guide posted in room 150 (see Appendix A). Number of
> plates refers to the number of plates in the in stack.*
>
> ***NOTE 2:** Always load production plates in fw/rv pairs on the same
> instrument. This means that plates can only be loaded in even numbers.
> If the loading guide calls for an odd number of plates, load that
> number plus one plate so that pairs are not split.*

2.  Remove the DNA sample plates with the oldest BET date from the 4°C
    refrigerator in Rm141 labeled "ABI3730 Backlog."

> ***NOTE 1:** Shuffle the plates so that not all of the plates from the
> same BET date or library are loaded onto the same ABI.*
>
> ***NOTE 2:** Always load 5 pairs of plates from the most current BET
> date on 5 different instruments. These plates should be loaded to the
> bottom of the in stack.*

3.  Quick spin the sample plates in a centrifuge at **1 minute at
    1,000rpm**.

4.  While keeping the forward/reverse pairs together, assemble the
    plates into gray bases with the white retainer tops. Make sure that
    the tray is fully assembled and that the retainer clips on either
    side are fully snapped into the base.

> ***NOTE:** If the tray is not properly assembled, the sequencer will
> not be able to run the plate.*

**3. [ABI 3730xl Setup & DNA Sample Loading]{.underline}**

> ***NOTE:** See* Spring 2005 Instrument Configurations *for sample
> plate loading matrix*

1.  Start the Instrument:

> ***NOTE:** In most cases, the instrument will be running. Sample
> loading will begin at step **d** below.*

a.  On the instrument, ensure that the:

    i.  Oven door is closed.

    ii. Instrument door is closed.

    iii. Stacker Drawer is closed.

    iv. Buffer, water, and waste trays are loaded.

b.  Make sure that the computer is on and logged in as the proper ABI
    instrument.

c.  If the instrument is not on, press the on/off power button located
    on the front of the instrument. Ensure that the green instrument
    status light is on and constant before proceeding (this takes about
    1 minute).

    i.  The light on the left hand side is the instrument status light.
        It is solid green when the instrument is "ready," it blinks
        orange when the instrument is initializing, and it blinks red
        when there has been an instrument failure.

    ii. The light on the right hand side is the stacker light. It is
        solid green when the stacker door is closed and blinks green
        when the stacker door is open.

d.  Prepare the Instrument and Loading DNA Sample Tray Assemblies
    (standard 3kb and 8 kb production plates)

> ***NOTE 1:** 3kb and 8 kb sample plates will be stored in bins in the
> ABI Backlog labeled "1/32 BigDye," "1/16^th^ BigDye," or "1/10^th^
> BigDye"*
>
> ***NOTE 2:** See* **Spring 2005 Instrument Configurations** *for a
> list of standard 1/32^nd^ 1/16^th^ and 1/10^th^ dedicated instruments.
> All instruments run the standard **15S** protocol, but the varying
> BigDye concentration plates are assigned to specific instruments to
> monitor the affect of the different chemistries on array performance.*

i.  Make sure that the instrument's green status light is on (either
    flashing or constant).

ii. Make sure that adequate levels of buffer and water are in the
    appropriate reservoirs.

iii. Check the level of POP-7 polymer in the bottle to ensure sufficient
     volume for run.

iv. Scan in the generic ABI barcode on the side of the DNA sample plate
    into the Sample Sheet Manager.

> ***Important!** Make sure that it is successfully imported into the
> Sample Sheet Manager and that the proper number of quadrants is
> selected at the scan step.*

v.  Pull open the stacker drawer. The stacker light flashes green.

vi. Open the metal door of the In Stacker tower and place up to 16
    (maximum) tray assemblies into the stacker (refer to the loading
    guide posted in room 150 or see Appendix A).

> ***Important!** Ensure that the tray assembly is fully assembled (both
> retainer clips are fully fastened to the base) and that it sits flat
> in the In Stacker. Failure to do so may result in instrument crash*.
>
> ***NOTE***: *The bottom tray will run first.*

vii. Close the metal In Stacker door and the stacker drawer. The stacker
     light becomes a constant green.

> **NOTE:** If the instrument is not already running a plate, then once
> the plates are loaded into the In Stacker and the Stacker drawer is
> closed, "Unknown" appears in the Data Collection Viewer's Input Stack
> window and the "Play" button on the top toolbar turns green.

viii. If the instrument is currently running when you add plates to the
      In Stacker, there is nothing left to do. The instrument will
      automatically run plates until the In Stacker is empty. If the
      instrument is not processing plates, click on the green **RUN**
      button.

ix. Click on the **OK** button to start processing plates.

```{=html}
<!-- -->
```
e.  Prepare the Instrument and Loading DNA Sample Tray Assemblies
    (Fosmid plates)

> ***NOTE 1:** Fosmid plates will be stored in the ABI Backlog in a
> specially marked "Fosmid" bin. The JGI barcode will be marked with a
> vertical green stripe.*
>
> ***NOTE 2:** certain instruments are near-dedicated instruments for
> processing Fosmid plates. These instruments have a modified run
> conditioned installed as their default for processing Fosmid plates.
> Fosmid plates should be loaded exclusively on these instruments unless
> additional sequencing capacity is required. See* Spring 2005
> Instrument Configurations *for a list of fosmid dedicated
> instruments.*
>
> ***NOTE 3:** Fosmid plates should never be loaded on an instrument
> that has 3 or more sample plates already loaded unless the Fosmid
> plates are loaded to the bottom of the in stack.*

i.  Follow steps **i-ix** of **d. Prepare the Instrument and Loading DNA
    Sample Tray Assemblies** above.

```{=html}
<!-- -->
```
f.  Prepare the Instrument and Loading DNA Sample Tray Assemblies
    (standard 3kb and 8kb production plates on Fosmid-dedicated
    instruments)

> ***NOTE:** Standard production plates should only be loaded on
> fosmid-dedicated instruments if there are not enough Fosmid plates
> available to keep these instrument running continuously.*

i.  Follow steps i-iv of **d. Prepare the Instrument and Loading DNA
    Sample Tray Assemblies** above.

ii. After scanning in the sample plate, select the **Plate Manager**
    link from the left hand frame of the Unified Data Collection
    software screen.

iii. Scan the generic barcode into the **Scan or Type plate ID** field.

iv. The plate name will appear in the field below. Select the plate and
    hit the **Edit** key on the bottom of the screen. The **Sequencing
    Analysis Plate Editor** window will appear.

v.  Select the cell found at row A1 under the column heading
    **Instrument Protocol**. A drop down menu will appear. Select the
    **15S** instrument protocol.

vi. Click on the column heading titled **Instrument Protocol** to select
    the entire column. Hit **Ctrl+D** on the keyboard. This will change
    the Instrument Protocol for the entire row to **15S**.

vii. Follow steps **v-ix** of **d**. Prepare the Instrument and Loading
     DNA Sample Tray Assemblies above.

```{=html}
<!-- -->
```
g.  Prepare the Instrument and Loading DNA Sample Tray Assemblies
    (Fosmid plates on standard 1/16^th^ or 1/32^nd^ instruments)

> ***NOTE 1:** Fosmid plates should only be loaded on 1/32^nd^ or
> 1/16^th^ instruments if there are too many Fosmids to process on the
> near dedicated instruments.*
>
> ***NOTE 2:** Fosmid plates should never be loaded on an instrument
> that has 3 or more sample plates already loaded unless the Fosmid
> plates are loaded to the bottom of the in stack.*

i.  Follow steps **i-iv** of **f. Prepare the Instrument and Loading DNA
    Sample Tray Assemblies (standard production plates)** above.

ii. Select the cell found at row A1 under the column heading
    **Instrument Protocol**. A drop down menu will appear. Select the
    **FOS** instrument protocol.

iii. Click on the column heading titled **Instrument Protocol** to
     select the entire column. Hit **Ctrl+D** on the keyboard. This will
     change the Instrument Protocol for the entire row to **FOS**.

iv. Follow steps v-ix of d. Prepare the Instrument and Loading DNA
    Sample Tray Assemblies above.

```{=html}
<!-- -->
```
h.  Ensure that the Out Stack is routinely emptied of processed trays
    (once per day) so that it will not overflow and stop the instrument.

i.  Once the plates have been removed from the Out Stack, go to the
    Venonat database, click on **ABI3730 DB**  [**Scan to Trash (Using
    ABI
    Barcodes)**](http://venonat.jgi-psf.org/psf/CAPGROUP.FORM_SCAN_TO_TRASH_NEW.show)
    form  scan in the plates' generic barcodes to verify that all
    plates have been fully processed.

> ***NOTE:** If a plate has not been processed, seal it with a clear
> seal and place it in the bin labeled "Plates not posting" in the deli
> refrigerator in room 147. Plates may take up to one hour to post.*

**4. [Reagent swaps required for ABI 3730xl maintenance]{.underline}**

> The ABI 3730xl contains fluid reagents that must be replenished
> regularly. These reagents include 1X Buffer with EDTA solution,
> Milli-Q water, and POP-7 polymer (Refer to **Reagent/Stock
> Preparation** for instructions on preparation of the 1X buffer
> solution). In order to replace the reagents, all instruments must
> first be paused. Reagent Swaps occur on Monday, Wednesday, and Friday
> of each week.

1.  Select the yellow pause button from the interface on the **Unified
    Data Collection** screen. Instruments that have 30 minutes of run
    time remaining or less should be paused.

> ***NOTE 1:** Operators only pause instruments with 30 minutes or less
> of run time to reduce the amount of time that instruments sit idle.
> This is more efficient than pausing an entire room of sequencers.*
>
> ***NOTE 2:** At the conclusion of their current runs, the instruments
> will pause with the sample plate held in the park position. The
> instrument will automatically maintain the oven and cell heater to
> their desired temperatures.*

2.  While waiting for the instruments to come to their idle state,
    remove the 1X running buffer as well as the POP-7 from the 4^o^C
    deli in Rm149 and allow them to come to room temperature.

> ***NOTE:** Installing cool running buffer can hamper instrument
> performance, while installing cool polymer can lead to the
> introduction of bubbles and result in run failures!*

3.  There are extra labeled water and waste tanks for each instrument.
    Fill each tank with **80ml** of Milli-Q water (so that the top of
    the meniscus is at the black line) and place the tank cap with the
    96-well septa cover on the tank.

4.  Fill the extra, unlabeled buffer tanks with **80ml** of buffer to
    the dark fill line on the side of the container and place the tank
    cap with the 96-well septa cover on the tank.

> ***Important!** Make sure that the exterior surface of the buffer tank
> is dry, and wipe it down with a paper towel if necessary. Moisture on
> the buffer tank can lead to arcing! Do not fill these tanks with water
> instead of buffer. Doing so may result in instantaneous array
> failure!*

5.  Set a buffer, water, and waste tank by each instrument.

6.  Change the buffer, water and waste tanks as the instruments complete
    their runs:

    a.  Once the green indicator light is a solid green, push the "tray"
        button on the front panel of the sequencer. This will move the
        buffer tank from the capillary position to the buffer station.

    b.  Open the door and remove the buffer, water and waste trays.

    c.  Open the trays by unclipping the white retainer tops and
        removing them from the black tray bottom.

    d.  Remove the used tanks and set them to the side.

    e.  Install a fresh tank in each tray and replace the retainer top.

    f.  Place each tray in its appropriate position on the ABI 3730
        instrument (*Refer to Figure 1 in Appendix A*).

        i.  Place the buffer tank in the far left station.

> ***Important!** Do not spill buffer onto the gripper arm when
> returning the buffer tray. This can lead to shortages and severely
> damage the autosampler!*
>
> ***NOTE:** Make sure that the cord attached to the buffer tray does
> not interfere with the gripper.*

ii. Place the water in the middle station.

iii. Place the waste in the far right station.

iv. Make sure that the retainer tops are securely in place and that they
    are properly aligned with the septa holes. Failure to properly
    replace the retainer cap can result in bent or broken capillaries.

```{=html}
<!-- -->
```
7.  Take the used buffer, water and waste tanks to the sink.

8.  Replace the buffer in the buffer bottle:

    a.  Take each instrument's buffer bottle to the sink and empty the
        buffer.

    b.  Rinse the bottle with Milli-Q water and wipe the bottle with a
        paper towel to quickly dry it.

    c.  Fill the buffer bottle with **67ml** of 1X running buffer (so
        that the meniscus is at the red line).

    d.  Return to the instrument with the filled buffer bottle.

    e.  Replace the buffer bottle by sliding it around the O-ring and
        onto the lower block.

    f.  Once the bottle is secure, rotate it so that the escape hole is
        facing towards you.

```{=html}
<!-- -->
```
9.  Replacing the POP- 7 polymer required for ABI 3730xl weekly
    maintenance:

> Polymer should be changed approximately once per week. Each polymer
> bottle should be labeled with tape with the date of installation
> written on it. Do not write directly onto the polymer bottle itself.

a.  Before installation, the appropriate polymer bottle must be assigned
    to the instrument in the database:

b.  In the Venonat database, select [**ABI3730
    DB**](http://venonat.jgi-psf.org/psfs/psf.wwv_main.main?p_language=us&p_cornerid=6639&p_currcornerid=1&p_full=1)
     [**Polymer and Array
    tracking**](http://sunbeam.jgi-psf.org:8080/ABI/arrayPolymerChange.vm)
     select the appropriate sequencer from the drop down menu and press
    the **Select Sequencer** button. On the bottom of the page, scan the
    lot number into the appropriate tab.

**NOTE 1:** Be sure to scan the lot number and not the part number or
the expiration date, as the barcode scanners are hard-carriage delimited
and will submit the selection immediately upon scanning. Do not type the
lot number, and avoid inserting spaces into the field before scanning,
as any typos or extra characters will compromise the ability of the
database to accurately track polymer lot performance.

**NOTE 2:** If the barcode does not scan in, type the lot number
manually. Inform the lead operator.

c.  If the lot that is being installed is different from the lot of the
    previous bottle, the sample plates in the In Stack must be rescanned
    with the sample sheet manager. In order to decrease instrument
    downtime, this step can be performed on sequencers as needed once
    all reagent swaps have been completed. This will re-assign the plate
    with the appropriate polymer lot. The plates do not need to be
    deleted from the Plate Manager at this step.

> ***NOTE:** Plates must be scanned one at a time for this step. The
> Sample Sheet Manager will display an error message that states that
> the "plate already exists in the database." This error message has to
> do with the importation of the sample sheet to the UDC software and
> can be ignored at this time, as it will not prevent the sample plate
> from processing.*

d.  After the water, waste, and buffer tanks have been swapped, replace
    the POP-7 polymer bottles:

```{=html}
<!-- -->
```
i.  Remove the used polymer bottle by gently unscrewing it from the lid
    attached to the pump block.

ii. Unscrew the fresh polymer bottle and gently feed the tube into the
    polymer, being careful not to introduce any bubbles.

iii. Fasten the lid and place the polymer bottle into the plastic
     holder.

iv. Turn the polymer bottle so that you can monitor the level of polymer
    and date on tape.

v.  Do not introduce any kinks into the tubing.

vi. Press the yellow and green **RESUME** button to unpause the
    instrument.

> ***NOTE:** Be sure the instrument status light is solid green before
> resuming plate processing.*

e.  Replace the unused buffer and POP-7 polymer in the 4^o^C deli in
    Rm149.

**5. [Cleaning Buffer, Water, and Waste Tanks]{.underline}**

1.  In the sink in Rm150, stagger the waste reservoir caps with septa
    and run hot tap water over the topmost cap for at least 5 minutes
    (*Refer to Figure 2 in Appendix A*).

2.  Rinse each cap with Milli-Q water separately.

3.  Shake off the residual water and place the caps upside down on a
    paper towel in a staggered position to dry (*Refer to Figure 3 in
    Appendix A)*.

4.  Repeat steps **5.1 -- 5.3** for the waste tanks.

***NOTE 1:** Do not mix tank types in the sink while washing.*

***NOTE 2:** Make sure that all of the dried polymer is removed from the
sides of the tank.*

5.  Repeat steps **5.1 -- 5.4** for the water caps and tanks.

6.  Repeat steps **5.1 -- 5.4** for the buffer caps and tanks with the
    exception that the cleaning will be performed in Rm122.

# Reagent/Stock Preparation

**[1X Running Buffer]{.underline}**

***NOTE:** Buffer can be made on Tuesdays, Thursdays, and Saturdays*

1.  Add **200ml** of 10X ABI 3730 Buffer with EDTA to a 2-L bottle.

2.  Add **1.8L** of Milli-Q water to the 2-L bottle.

3.  Mix well.

4.  The 1X running buffer can be stored at 2 to 8ºC for up to 1 month.

5.  Label the bottle: *Lot #, Date, and your initials*.

6.  Just over 5 buffer bottles are required for a single reagent swap.
    No more than 15 bottles of 1X running buffer should be prepared on
    any single day.

**[Standard Dilution for Spectral Calibration]{.underline}**

***NOTE:** Spectral standard to be prepared on an as needed basis.*

1.  Remove 1 unprepared BigDye v3.1 Seq. Std. Tube from the -20°C
    freezer and allow it to thaw at room temperature.

2.  Remove Hi-Di Formamide from the -20°C freezer and thaw at room
    temperature.

***Important!** Always wear gloves when handling formamide or solutions
containing formamide.*

3.  Spin down the thawed unprepared BigDye v3.1 Seq. Std. Tube for **30
    seconds at 6,500rpm**.

4.  Add **1.5ml** of thawed Hi-Di Formamide to the BigDye v3.1 Seq. Std.
    Tube.

5.  Vortex the tube for one minute.

6.  Place contents into a Matrix Reagent Reservoir

7.  Fill to **125μl** using a 12-channel Matrix pipette.

8.  Dispense **5μl** into only the 1^st^ quadrant of an ABI plate.

9.  Purge the remaining contents back into the Matrix Reagent Reservoir.

10. Return the remaining contents into the tube using a 1000-μl Rainin
    Pipet Lite.

11. Store at -20°C for future use.

12. Spin down the plate in an Eppendorf centrifuge at **1 minute at
    1,000rpm**.

13. Seal the plate using a foil seal only.

# Instrument Maintenance

**Monthly Maintenance:**

Preventative maintenance (performed by the user) is required for each
sequencer on a monthly basis. Operator time is dedicated for performing
maintenance in each shift schedule. Maintenance can be performed
concurrently on up to 4 instruments at a time. Maintenance is to be
performed on the sequencers in numerical order (from ABI201 to ABI268).

**1. [Recording Values and Initial Inspection.]{.underline}**

1.  Identify the sequencers you will be performing maintenance on.
    Select the yellow "Pause After Completion of Current Run" button
    from the interface of the **Unified Data Collection Screen**. Pause
    only instruments with less than one hour of run time remaining.
    Pause no more than three instruments at a time.

2.  Prior to the completion of the current run, on the left hand side of
    the **Unified Data Collection** screen, select the **Instrument
    Status** link:

    a.  Use *Table 1* in **Appendix** **A** section to note the
        *Electrophoresis voltage, the Laser Current, and the Cell Heater
        Temperature*.

    b.  In order to complete the **Fill Time** field:

        i.  Select **Event Log** for active runs from the left hand from
            of the **Unified Data Collection** screen.

        ii. Scroll through the last four complete sample runs. Determine
            the amount of time that elapsed between the **Filling Array
            with Polymer** command and the **Rinsing Capillary Tips**
            command.

        iii. Determine the average of these four fill times in minutes
             and seconds.

    c.  Record the values under **OCTOPUS\\QC\\QC
        Shared\\ABIdata\\Maintenance** in the Excel spreadsheet titled
        **Maintenance Recorded Values**.

3.  Once the run has completed, open the main door and use a Kimwipe or
    paper towel dampened with Milli-Q water to wipe down the internal
    and external surfaces of the instrument, including the in and out
    stack doors, the polymer block, the computer screen, the mouse, and
    the barcode scanner.

4.  Remove the sample plate from the park position. If all four
    quadrants have completed processing, leave the sample plate out for
    debasing. If the plate processing is incomplete, place the sample
    plate at the bottom of the in stack.

5.  Open the oven door. As you open the door, observe the three fans
    above the array chamber. Ensure that all three fans are spinning as
    you open the door (these fans will slow to a stop after the door is
    opened).

> ***NOTE:** If any fan is not spinning, immediately contact an Applied
> Biosystems service engineer. Open a service call by sending an email
> to the service call center at:
> servicecallcenter@appliedbiosystems.com.*

6.  Check for any evidence of polymer leakage surrounding the detection
    cell cover. If any leakage is present, alert the ABI Lead Operator.

> ***NOTE:** Do not open the detection cell cover! This can cause
> movement of the capillary array detection cell.*

7.  Check the stacker drawer itself as well as the hinged metal doors.
    Be sure that everything moves properly. Inspect the thin metal arm
    that extends from the interior surface of the stacker door to be
    sure that it is not bent or misshapen. Also be sure that the main
    instrument door is properly aligned and closes easily.

> ***NOTE:** If there are any mechanical problems with the door, contact
> an Applied Biosystems service engineer. Open a service call by sending
> an email to the service call center at:
> servicecallcenter@appliedbiosystems.com*

8.  Remove the drip pan as well as the buffer, water and waste tank
    holders and rinse these in the sink with hot water followed by
    Milli-Q water. Dry before replacing. When replacing the buffer tank,
    be sure that the cord will not obstruct the movement of the
    autosampler.

9.  Make sure that the plastic capillary container is present in the
    drip pan, as well as the capillary tip bottle and the window surface
    cover. Check to make sure that the buffer, water, and waste labels
    are present and in their correct locations.

```{=html}
<!-- -->
```
1.  **[Check for Disk Errors]{.underline}**

    1.  Right click on **My Computer**.

    2.  Click on **Manage**.

    3.  Click on **Event Viewer**.

    4.  Click on **System**.

    5.  Check for red errors with disk as its source.

```{=html}
<!-- -->
```
3.  **[Flushing the PDP Water Trap]{.underline}**

    1.  Fill the 20-ml syringe (supplied with the Polymer Block Cleaning
        Kit item 4335860) with 5-10ml Milli-Q water. Attach the syringe
        to the forward facing Luer fitting by threading the syringe onto
        the fitting.

    2.  Loosen the fitting by one-half turn counter clockwise. Open the
        exit fitting on the side of the pump block by turning it
        one-half turn counter clockwise.

    3.  Slowly apply force to the syringe to force water through the
        trap at a rate of approximately 5ml per 30 seconds. Flush 5-10
        ml of water through the water trap, catching the dripping water
        from the exit fitting in a 36 ml buffer bottle. Once this is
        complete, tighten the Luer fitting followed by the exit fitting
        and remove the syringe. Dry the exterior surface of the polymer
        block if necessary.

4.  **[Completing the Water Wash]{.underline}**

    1.  Take an old bottle of polymer, and rinse it out completely with
        Milli-Q water. Once you are sure that the bottle is empty, fill
        it with Milli-Q water and replace the polymer bottle currently
        in use with the bottle now filled with water. Do not discard the
        currently installed polymer bottle, as it will be replaced at
        the completion of the water wash.

> ***Important!** Always wear gloves when handling POP-7!*

2.  Remove the **67 ml** buffer bottle from the lower block and replace
    it with an empty **36 ml** bottle. This will collect the water from
    the water wash. There is no need to discard the buffer in the **67
    ml** bottle.

3.  Restart the Viewer and Instrument Service on the Service Console to
    access the Wizards menu.

4.  Run the Water Wash Wizard by selecting **Wizards**  **Water Wash
    Wizard** from the dropdown toolbar on the top of the **Unified Data
    Collection** interface and follow all instructions (this will take
    approximately five minutes).

> ***NOTE:** The water wash wizard will flush water through the entire
> Polymer Delivery Pump assembly. This is done to clean the valves and
> prevent leaking. At the completion of the water wash, you will be
> prompted to prime the block with polymer.*

5.  **[Ambient Temperature Check]{.underline}**

    1.  Using the thermocouple thermometer, record the ambient
        temperature directly in front of the ABI.

6.  **[Oven Temperature Check]{.underline}**

    1.  Open the instrument door and oven door.

    2.  Take the probe end of the thermocouple and feed it through the
        "bottle holder" of the installed capillary array so that the
        probe is near the capillaries.

    3.  Close the oven door.

    4.  Monitor the **Instrument Status** window. Record the temperature
        on the thermocouple monitor once the oven temperature has
        stabilized at 60^o^C.

    5.  Open the oven door and remove the thermocouple

7.  **[Cell Heater Temperature Check]{.underline}**

    1.  Open the **Detection Cell** cover.

    2.  Insert the probe end of the thermocouple so that the
        thermocouple is positioned against the detection cell just below
        the capillaries.

    3.  Close the **Detection Cell cover** as well as the **Oven Door**.

    4.  Monitor the **Instrument Status** window. Record the temperature
        on the thermocouple monitor once the cell heater temperature
        stabilizes to the temperature recorded in **1.2**.

    5.  Open the **Detection Cell cover** and remove the thermocouple.

    6.  Close the **Detection Cell cover**, the **Oven Door**, and the
        **Main Door**.

8.  **[Spatial Calibration]{.underline}**

    1.  Check the capillary tips to ensure that they are not crushed or
        damaged.

    2.  Perform a spatial calibration by selecting **Spatial Run
        Scheduler** from the menu on the left hand side of the **Unified
        Data Collection** screen and selecting **spatial_fill** (this
        option includes a polymer fill) from the drop down menu.

    3.  After the data collection is completed, be sure that all of the
        peaks have absolute values of approximately 1000 rfu's.

    4.  Verify that the yellow cursors defining the capillary positions
        are located on the tops of the peaks.

    5.  Check the left right spacing to be sure that the values are all
        between 4 and 8 (with 5 being ideal).

    6.  Verify that the pixel positions have not changed from the
        original run (if the detection cell has not been moved), paying
        special attention to the first 3 and last 3 pixel positions.

        a.  Select the **Run History** link from the left hand frame of
            the **Unified Data Collection** interface. Expand the menu
            options by clicking the "+" sign next to the **Run History**
            link.

        b.  On the right hand frame, select **Advanced Search** from the
            "Type of Search" drop down menu.

        c.  A grid will appear. Select the "Condition" field next of the
            "Run Name" category with the mouse. A drop down menu will
            appear. Select "contains."

        d.  In the adjacent "Value 1" field, type in **yesterday's**
            date in the following format: December 1, 2003 would be
            entered as **2004-12-01**. Click the search button. A list
            of runs will appear in the field below the selection grid.

        e.  Toggle between the **Spatial Calibration Viewer** (which
            displays the original spatial calibration) under **Run
            History** and the **Spatial Run Scheduler** (which displays
            the newly acquired calibration) to compare the pixel
            positions.

        f.  If the pixel positions have remained unchanged, click the
            **Accept Spatial** button on the **Spatial Run Scheduler**
            screen.

    7.  If these conditions are not met:

        a.  Take a screenshot (alt +Print Screen).

        b.  Save the screenshot of the calibration in the **Bad
            Spatial** folder on the Miracle D drive.

        c.  Reject the spatial by clicking the **Reject Spatial** button

        d.  Take no further action.

> ***NOTE:** The instrument will continue to run with its original
> spatial calibration values. However, this information is extremely
> valuable for Instrument/Array QC purposes.*

9.  **[Instrument Ambient Temperature Check]{.underline}**

    1.  Select **EPT Viewer** under **Event History**.

    2.  Using the mouse, zoom in on the **Ambient Temperature** line on
        the line graph.

    3.  Record the approximate ambient temperature at the end of the
        run.

10. **[Database Check and Instrument Restart]{.underline}**

    1.  Once the current run has completed, select the **Database
        Manager** link from the left hand side of the **Unified Data
        Collection** Screen to check the database manager to verify that
        the database is below 50% of its capacity.

> ***NOTE 1:** If it is above 50%, **verify that there is no need to
> re-extract any runs** (due to machine errors or for experimental
> purposes, check daily update emails!). Resolve any plate transferring
> errors at this time if needed (see **ABI 3730xl Data Transfer Error
> Resolution** SOP). Wait for the completion of the current run and be
> sure that all 96 lanes extract correctly. Clear the database by
> selecting **Clear Processed Plates** and proceed with maintenance.*

2.  If after clearing the database the % full does not change:

> ***NOTE:** The following instruments have exhibited this behavior in
> the past and may continue to do so in the future: RNDABI1, ABI206,
> ABI211, ABI217, ABI218, ABI221, ABI231, and ABI252.*

a.  Right click the **Instrument Service** square of the Service Console
    and select **stop**. Wait for the **Viewer** and **Instrument
    Service** consoles to become red squares.

b.  Navigate to : **E:\\AppliedBiosystems\\UDC\\DataCollection\\bin**.

c.  Double click **dc_export_database.bat**. A DOS screen will appear
    and will remain open for several minutes.

d.  Once the DOS screen has disappeared, return to the **bin** directory
    and double click **dc_import_database.bat**. A DOS screen will
    appear and will remain open for several minutes.

e.  Once the DOS screen has disappeared, right click the red **Viewer**
    square in the **Service Console** window and select **start**.

f.  The viewer will re launch. Return to the **Database Manager** to
    ensure that the % full has been reset.

```{=html}
<!-- -->
```
3.  Stop all four components of the Service Console and power down the
    sequencer. Restart the PC. After logging in, restart Unified Data
    Collection as well as the Sample Sheet Manager program. Power up the
    sequencer and resume processing sample as normal (Refer to the
    **Procedure** section).

    a.  Occasional the UDC components will not restart properly. If one
        of the modules freezes (does not go to a green square), if the
        viewer will not launch, or if an error appears stating "Maximum
        concurrent number of User's has been reached," restart both the
        PC and the Sequencer.

    b.  If errors persist, submit open a **Service Call** (see
        **Instrument Restart Guide**).

4.  Check the status lights on the UPS connected the sequencer and PC.
    Enter an instrumentation work request if any of the status lights
    are red.

5.  Properly position the monitor arm so that the screen is level and
    parallel to the front of the instrument. If the monitor arm cannot
    be adjusted easily, enter an instrumentation work request.

```{=html}
<!-- -->
```
11. **[Peak 1/Base Spacing Determination]{.underline}**

    1.  On the sequencer, double click the **Sequence Analysis v5.1**
        icon located on the desktop.

    2.  Login using the password.

    3.  Navigate to the **RunName** folder located at
        **E:/AppliedBiosystems/UDC/DataCollection/Data/InstrumentType/InstrumentName/RunName**.

    4.  Open the **xfr** folder for the last completed run.

    5.  Choose **File -\> Select All** to select all samples in the
        folder.

    6.  Right click the select files and choose **Open**. The files will
        appear in **Sequence Analysis v5.1** window.

    7.  Highlight the two columns named **Peak 1** and **Spacing** once
        all 96 sample files have been imported. Select **File -\>**
        **Copy** to copy all of the files.

    8.  Paste the values into an excel spreadsheet and determine the
        values for each column.

    9.  Record these values.

# SOP Approval

  ----------------------- ----------------------- -----------------------
  **DEPARTMENT**          **APPROVED BY**         **DATE**

  **Lab Supervisor**                              

  **Research &                                    
  Development**                                   

  **Instrumentation**                             

  **QC**                                          

  **Purchasing**                                  

  **EH & S**                                      

  **Informatics**                                 

  **Seq Assessment &                              
  Analysis**                                      

  **Dept Head of Prod                             
  Seq**                                           
  ----------------------- ----------------------- -----------------------

# Appendix A

**[FIGURES]{.underline}**

Figure 1. Buffer, water and waste tanks loaded onto the ABI 3730xl
instrument. ![](media/image1.jpeg){width="5.052777777777778in"
height="3.8in"}

Figure 2. Running hot tap water over staggered reservoir caps in the
sink.

![](media/image2.jpeg){width="2.3805555555555555in"
height="3.167361111111111in"}

Figure 3. The caps and tanks placed down on a paper towel in a staggered
position to dry.

![](media/image3.jpeg){width="3.1805555555555554in"
height="2.390277777777778in"}

![](media/image4.wmf)

**Loading Guide -- Monday thru Thursday**

Plates Required to Run until 9 am next day

  ----------------------------------- -----------------------------------
  9 am                                4

  11 am                               4

  1 pm                                3

  3 pm                                3

  5 pm                                3

  7 pm                                3

  9 pm                                2
  ----------------------------------- -----------------------------------

**Loading Guide -- Fridays**

*Plates Required to Run until 9 am Monday*

  ----------------------------------- -----------------------------------
  9 am                                11

  11 am                               11

  1 pm                                10

  3 pm                                10

  5 pm                                10

  7 pm                                9

  9 pm                                9
  ----------------------------------- -----------------------------------

Loading Matrix By Sequencer and Insert Size (Chemistry Type)

  ------------------------------ ------------ ------------ ---------------
                                                           

                                                           

                                                           
  ------------------------------ ------------ ------------ ---------------

# Appendix B

**[AUDIT TRACKING]{.underline}**

**5/26/04 -- 5/27/04** -- Danielle Mihalkanin and Anna Ustaszewska
performed an audit between 10:00AM (5/26) -- 11:00AM (5/27). The
operators were Jen Alvernaz, Cynthia Maunes, and Damon Tighe.