MegaBACE 4500 DNA Analysis System

**Users Protocol**

> Version Number: 1.2 (Appended Ver2.4 of MB4000 User SOP)
>
> Production Start Date: 11/11/04
>
> Version 1.2 Date: 05/04/05
>
> Author: Chris Daum
>
> Reviewed/Revised by:

# Summary

This protocol outlines the operation of the MegaBACE 4500 DNA Analysis
System for high throughput capillary electrophoresis of DNA samples.

# Materials & Reagents

+----------------------------+-------------------------+--------------+
| **[Materials/Reagent       | *                       | **[Stock     |
| s/Equipment]{.underline}** | *[Vendor]{.underline}** | Number]{.    |
|                            |                         | underline}** |
| *                          | VWR                     |              |
| [Disposables]{.underline}* |                         | 62409-074    |
|                            | Fisher Scientific       |              |
| 384-well Nunc Plate 120μl, |                         | 342800-0020  |
| Clear                      | Fisher Scientific       |              |
|                            |                         | 642005-0680  |
| 2.0ML Vial, Natural, PPCO  | Edge Biosystems         |              |
| Sterile, 1000/cs           |                         | 48461        |
|                            | GE Healthcare\*         |              |
| 2.0ML Vial, Blue, PPCO     |                         | 13575001     |
| Sterile, 1000/cs           | GE Healthcare\*         |              |
|                            |                         | \-           |
| Clear Adhesive Plate       | Millipore Super-Q       |              |
| Sealers                    | System                  | \-           |
|                            |                         |              |
| *[Reagents]{.underline}*   | GE Healthcare\*         | \-           |
|                            |                         |              |
| MegaBACE 4500 2.6% V2E LPA | Watson Marlow           | \-           |
|                            |                         |              |
| MegaBACE 10X Running       | VWR Scientific          | \-           |
| Buffer                     |                         |              |
|                            | VWR Scientific          | \-           |
| Milli-Q water              |                         |              |
|                            | Eppendorf               | \-           |
| *[Equipment]{.underline}*  |                         |              |
|                            |                         |              |
| MegaBACE 4500 Sequencer    |                         |              |
|                            |                         |              |
| Watson Marlow Pump         |                         |              |
|                            |                         |              |
| Heat Block                 |                         |              |
|                            |                         |              |
| Vortex                     |                         |              |
|                            |                         |              |
| Centrifuge 5810            |                         |              |
+----------------------------+-------------------------+--------------+

\* GE Healthcare, formerly Amersham Biosciences

# 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.

# Procedure

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

**1. [Pre-Run Setup]{.underline}**

1.  Verify the run start time using the posted Run Schedule in Rm120.

2.  Prior to the start time, assess how many instruments will be loaded
    and prepare the materials you will need for the run:

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a.  Check the sample freezer in Rm121 to see if plates have been
    assigned for the run. If no plates have been assigned, then pull
    plates from the sample freezer or BET Freezer201 in Rm140 and assign
    plates in FW & RV pairs to the same instrument for two runs.

b.  Ten minutes before loading sets of instruments, use the
    Watson-Marlow pump to fill the appropriate number of labeled
    translucent water tanks with **120ml** of Milli-Q water and labeled
    yellow buffer tanks with **120ml** of 1.5X running buffer.

> i\. To operate Watson-Marlow pump, press the **Start** button twice to
> begin priming the tubing and dispensing 120ml per cycle. To stop
> dispensing, press the **Stop** button once.
>
> ***NOTE:** The Watson-Marlow outlet tube is marked with lab tape.*
>
> ***Important!** Run 240ml additional Milli-Q water through the pump
> after running 1.5X buffer through it in order to cleanse it and
> prevent buffer residue buildup.*
>
> ***Important!** The dispense volume of the Watson-Marlow pump must be
> checked on a weekly basis. To do this, dispense into a graduated
> cylinder and measure the delivered volume; if it varies from 120ml
> [+]{.underline} 5ml, notify MegaBACE instrumentation so that the pump
> may be calibrated.*
>
> ***NOTE:** If the Watson-Marlow pump is non-functional, the water and
> buffer tanks may be manually filled using a graduated cylinder to
> measure out the 120ml dispense volumes.*

c.  Set out a single buffer tank and water tank for each instrument in
    the loading set.

**2. [Sequencing Run]{.underline}**

> ***NOTE:** A single sequencing run consists of loading sets of
> instruments. Refer to the posted Run Schedule for set information and
> start times. Make sure to fully complete the loading (matrix fill &
> sample injection) of one set before starting the next.*
>
> ***NOTE:** Refer to part 2 of Appendix B for an example of the loading
> scheme for instrument sets of 7.*

1.  Set out the matrix fill and pre-run materials you will need for each
    instrument in the set:

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a.  One buffer tank containing 120ml of 1.5X running buffer per
    instrument in the set.

b.  Six pre-spun (**2 minutes at 3,000rpm**) tubes of 2.6% V2E LPA per
    instrument in the set.

    1.  Perform the **Matrix Fill and Prerun** Protocol for a set of
        instruments:

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a.  In the **Instrument Control** window of the ICM software, with the
    **Matrix Fill and Prerun** protocol selected, click on **START**
    (perform this on each instrument in the set).

b.  ![](media/image1.png){width="0.375in"
    height="0.3020833333333333in"}Follow the instructions on the
    instrument display window to load the buffer tank into the left
    (cathode) side of each instrument followed by the matrix tubes into
    the right (anode) side of each instrument in the set; from the
    display window messages, verify that the drawers have been closed
    completely. *Refer to Figures 1 & 2 in Appendix A*.

> ***Important!** Check the matrix tubes for bubbles and make sure there
> are no empty tubes; do not load these unsuitable tubes. If empty tubes
> are found, set them aside and label them with the LPA lot# and date
> found. If bubbles are still present in any of the tubes, re-centrifuge
> them for 2 minutes at 3,000rpm.*

c.  After opening the anode and cathode drawers, wipe any spilled
    buffer, water or matrix with a damp paper towel.

d.  After loading the matrix tubes, listen for any high-pressure leaks.
    Inform MegaBACE instrumentation if any leaks occur.

    1.  Preparation of DNA samples for a set of instruments:

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a.  After loading the matrix tubes and while the instrument is
    performing its automated fill and prerun, prepare the DNA sample
    plates for the set of instruments being loaded.

    i.  Pull the assigned sample plates from the freezer and thaw them
        on the heat blocks for **15 seconds at 90^o^C**.

> ***NOTE:** Use provided timer for monitoring the thaw time of 15
> seconds.*

ii. Vortex the sample plates on the Vortexer for **2 minutes on setting
    7**; loading plates in stacks of no more than 4 high.

> ***NOTE:** Ensure that plates are secure in Vortexer, but do not
> over-tighten the top cover screw-clamps or put excessive pressure on
> the top cover itself.*

iii. Quick spin the sample plates in the centrifuge for **1 minute at
     1,000rpm**.

> ***Important!** Once the sample plates have been centrifuged they must
> be handled gently as not to induce bubbles or produce air pockets at
> the bottom of wells.*

b.  After preparing the DNA samples, the plates need to be scanned into
    the instrument on which they are to run; this will import the
    associated samplesheet record.

    i.  In the **Plate Setup** window of the ICM software, click on
        **NEW**.

    ii. In the **Plate ID** box, scan in the barcode on the sample plate
        that will be run on that instrument.

    iii. Verify that the correct values appear in the Electrophoresis
         Parameters: *Sample Injection Voltage: 2 KV, Run Voltage: 5.9
         KV, Sample Injection Time: 36 sec, Run Time: 215 min.*

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    1.  Perform the **Inject Samples and Run** Protocol for a set of
        instruments once the Matrix Fill and Prerun protocol has
        finished:

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a.  Set out the materials you will need for each instrument in the set:

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i.  One water tank containing 120ml of Milli-Q water.

ii. The prepared DNA sample plate assigned to each instrument.

iii. ![](media/image1.png){width="0.375in"
     height="0.3020833333333333in"}A 384-well NUNC Plate containing 85μl
     of 1.5X running buffer per well; place the plate on the Buffer
     Plate Adapter with well A1 located in the lower left-hand corner.

> ***Important!** Examine the buffer plate to ensure that each well has
> buffer in it and that the volume looks uniform across the plate.*

iv. ![](media/image1.png){width="0.375in"
    height="0.3020833333333333in"}6 pre-spun (**1 minute at 1,000rpm**)
    transparent blue micro-centrifuge tubes filled with 1.75ml of 1.5X
    running buffer.

> ***Important!** Examine the buffer tubes to ensure that each one has
> buffer in it, is bubble free, and that the volume looks uniform across
> all tubes.*

b.  In the **Instrument Control** window of the ICM software, with the
    **Inject Samples and Run** protocol selected, click on **START**.
    The **Select a Plate** window will appear, scan in the barcode on
    the assigned sample plate to continue (perform this on each
    instrument in the set).

> ***Important!** It is crucial to sample performance that the following
> steps are completed as quickly as possible. Otherwise, the quality of
> the sequencing run may be compromised.*

i.  Follow the instructions on the instrument displays to load the water
    tank into the left (cathode) side of each instrument in the set;
    from the display window message, verify that the drawer has been
    closed completely. *Refer to Figure 3 in Appendix A*.

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c.  Once the instruments finish their automated tip rinsing, the
    **Confirm to Continue** window will appear. The remaining steps will
    be performed on a subset of instruments at a time. *Refer to part 2
    of Appendix B for an example of the loading scheme of 7 instrument
    sets.*

    i.  Click **Enter** on the **Confirm to Continue** window on the
        first instrument of the subset.

    ii. Follow the instructions on the instrument display to load the
        assigned sample plate on the adapter into the left (cathode)
        side of the first instrument in the subset; from the display
        window message, verify that the drawer has been closed
        completely. *Refer to Figure 4 in Appendix A*.

> ![](media/image1.png){width="0.375in"
> height="0.3020833333333333in"}***Important!** Ensure that the clear
> plate seal is completely removed and that no torn pieces of the seal
> remain on the plate. Also ensure that the plate is placed level on the
> adapter. Remaining pieces of seal or improperly placed plates may harm
> the instrument if loaded.*

iii. Repeat steps i. & ii. for each individual instrument remaining in
     the subset.

> ***Important!** Do not click **Enter** on all of the instruments in
> the subset at once; steps i. & ii. must be done individually for each
> one before moving on to the next.*

iv. Once the sample injection has completed on the first instrument in
    the subset, the **Confirm to Continue** window will appear. Click
    **Continue** on the first instrument only.

v.  Follow the instructions on the instrument displays to load the 85μl
    1.5X buffer plate on the deep-well plate adapter into the left
    (cathode) side of the instrument followed by the blue 1.75ml 1.5X
    buffer tubes into the right (anode) side of the instrument. From the
    display window messages, verify that the drawers have been closed
    completely.

> ![](media/image1.png){width="0.375in"
> height="0.3020833333333333in"}***Important!** Examine the buffer plate
> and tubes before loading them into the instrument; ensure that the
> fill volumes are correct.*

vi. Repeat steps iv. & v. for each individual instrument remaining in
    the subset.

> ![](media/image1.png){width="0.375in"
> height="0.3020833333333333in"}***Important!** Do not click
> **Continue** on all of the instruments in the subset at once; steps
> iv. & vi. must be done individually for each one before moving on to
> the next.*

vii. Repeat steps i. -- vi. for all instruments in the second subset

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1.  Putting the instrument to sleep after the sequencing run (only to be
    done when instruments are to be slept for the night; refer to posted
    Run Schedule):

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a.  In the **Instrument Control** window, select the **Sleep
    Parameters** tab.

b.  In the **Sleep Time** field, enter the number of hours the
    instrument is to sleep (1-16 hours).

c.  Select the **Sleep After This Run** check box; the Instrument
    Control Manager will automatically store the capillaries in the run
    buffer tubes and buffer plate after the current run has finished.

> ***Important!** After the entered sleeping time has elapsed, it
> requires 3 full hours for the instrument to warm back up before it may
> be run again*.

**3. [Monitoring and Assessing the Quality of the Run]{.underline}**

1.  Check the Fluorescence Image Display area to ensure that the scan
    images are being collected across the plate:

    a.  Click on the **Run Image** window of each instrument.

    b.  Click on the well buttons above the Fluorescence Image Display
        to view electropherograms for individual lanes. Initially, all 4
        baselines should be present and at an intensity above zero. The
        lack of an image after 45 minutes (no sequencing data or
        baseline noise across all channels) may be indicative of
        instrument problems; notify MegaBACE instrumentation
        immediately.

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2.  Check the capillary current for uniform electrical current:

    a.  In the **Options** drop down menu select **Current Monitor**.

    b.  Examine the Current Monitor window for uniformity of current
        values across the plate; the values should be within a range of
        6.5 -- 8.5 μAmps. Check for regions of no current, abnormally
        high/low current, or erratic current that constantly changes
        values.

        i.  Individual zeros may indicate broken/clogged capillaries,
            empty buffer plate wells, or the presence of bubbles.

        ii. Groups of zeros or regions of abnormally high/low current
            may indicate more severe problems (i.e. broken capillaries
            or empty buffer tubes & empty buffer plate wells); notify
            MegaBACE instrumentation or administration immediately.

> ***NOTE:** Abnormal current values of individual capillaries may be
> referenced to the **Bad Cap** excel sheet located on the desktop of
> each instrument's PC.*

3.  While monitoring MegaBACE runs, if any abnormal electropherograms
    are found which may include overloading, bubbles in the capillaries,
    sequencing artifacts, tandem repeats, or double traces, take a
    screen shot, paste it onto a PowerPoint slide (include: *date, run
    #, instrument #, average intensity, the room's temperature, and a
    brief description of the abnormal sequence*) and email it.

4.  While loading and monitoring runs in the MegaBACE Sequencing Lab, it
    is important to take note of and report any aberrations from the
    normal workflow. With as much detail as possible (documentation and
    screen shots if applicable) notify MegaBACE administration and
    instrumentation of the abnormality and send out an electronic
    depiction. This would include any abnormalities, no matter how
    seemingly small, within the following areas:

    a.  MegaBACE Instruments -- on-screen errors; software/hardware
        crashes; instrument malfunctions; poor current; failed or
        blinking lasers; difficulty in opening anode/cathode drawers;
        operator error while loading; aborting of runs.

    b.  Sample Plates -- missing wells; damaged plates or barcodes;
        blank run images; run images with abnormal motifs.

    c.  Loading Runs -- instruments down for service or PMs; evaporated
        buffer plates when pulling them out of instruments; loading sets
        at a different time than what is posted on the posted schedule;
        loading plates on the wrong MegaBACE instrument.

    d.  Venonat Database -- outages; forms not working; scanning plates
        into the wrong MegaBACE field; choosing the wrong reagent lots,
        run number, or operator; plates that have not posted.

    e.  Reagents -- changing of lots; empty or partially filled matrix
        tubes; buffer plates with empty wells; supply inventory; low on
        supplies.

    f.  Supporting Equipment & Robotics -- issues with the
        Multidrop/Twister, Tecan, or Watson-Marlow pump; calibration
        checks; work requests.

    g.  Facilities -- room temperature; running of the auxiliary AC;
        issues with the sink or Super-Q.

**4. [Post-Run]{.underline}**

1.  Scan in the barcodes of the sample plates that were loaded into the
    MegaBACE database:

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a.  In the Venonat database, click on **MegaBACE DB**  [**Run Scan in
    Form
    MB4000s**](http://venonat.jgi-psf.org/WebDB/CAPGROUP.FORM_RUN_SCAN_IN_MB400.show)
     enter in: *run date, run #, your name as the operator, matrix lot,
    and buffer lot*.

b.  Click on **SUBMIT**.

c.  Scan in the barcodes of the sample plate corresponding to the
    MegaBACE instrument on which it was loaded.

d.  Click on **SUBMIT**.

    1.  The loaded sample plates will be stored for a two-week period
        before disposal:

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a.  After scanning the barcodes of the loaded sample plates into the
    Venonat database, tape the plates together and label with: *Run
    Date, Run Number, Operator's Initials.*

b.  Store the plates in the Loaded Samples freezer in Rm121, placing
    them on the shelf that corresponds to the day of the week that they
    were loaded.

c.  To dispose of plates after a two-week storage period:

    i.  In the Venonat database, click on **MegaBACE DB**  **Check
        Plate Status Before Disposing.**

    ii. Scan the barcodes of the plates slated for disposal into the
        **Enter Barcodes** box.

    iii. Check the selection boxes next to *Search for Yunian's QC
         Reports* and *Display only runs that are not posted*.

    iv. Click on **SUBMIT**.

    v.  An on-screen report will be generated, as long as a sample
        plate's name is [not]{.underline} displayed it may be thrown
        away in a lab garbage can. If a sample plate's name is
        displayed, the plate has not yet posted. Save these plates and
        notify MegaBACE administration.

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    1.  After loading the sets for a given run, the remaining water and
        buffer in the tanks used for the sequencing run must be disposed
        of and the tanks washed:

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a.  Take the water tanks and buffer tanks to the sink.

b.  For Water Tanks:

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i.  Dump and shake out the water into the sink and rinse the tanks with
    Milli-Q water only.

ii. Shake out the excess Milli-Q water and set the tank on the
    designated rack to dry.

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c.  For Buffer Tanks:

    i.  Disposal of LPA/Buffer mixture -- Turn on the sink's hot tap
        water and allow it to run down the drain. Once the water is hot,
        dump and shake out each buffer tank into the sink.

    ii. Rinse each buffer tank with hot tap water to remove all of the
        remaining LPA/Buffer mixture and shake out the excess water.
        Once visible LPA strings no longer appear while shaking, rinse
        the tanks with Milli-Q water. Shake out the excess water and set
        the tank on the designated rack to dry.

    iii. After rinsing out the buffer tanks, allow the hot tap water to
         run for at least 2 minutes to prevent drain clogs from forming.

**5. [Aborting a Running Protocol]{.underline}**

> In the event of an instrument issue (i.e. on screen error messages,
> abnormal or extreme current indicating an empty buffer plate or matrix
> tube, laser shut downs, operator error, etc.), the following steps
> will abort a run:

1.  In the **Instrument Control** window, click on **STOP** to end the
    running protocol. It will take the instrument \~30 seconds to abort
    the run and re-initialize itself.

> ***NOTE:** Should the **Matrix Fill & Prerun** protocol need to be
> aborted during the high-pressure fill, clicking **STOP** will not
> abort the protocol immediately; in order to abort immediately the
> instrument must be powered off using the power switch located on the
> right-lower side of the instrument.*

2.  If the issue is not serious in nature, the protocol may be restarted
    or the instrument may be left idle until the next run is ready to
    start. If there is not another run to start or the instrument
    requires service, it must be stored in water to prevent the
    capillaries from drying out (refer to **Section 6**).

3.  If a run is aborted and not immediately restarted, the prepped
    sample plate that was assigned to run will need to be re-queued.

    a.  In the Venonat database click on **MegaBACE DB**  [**Scan
        in**](http://venonat.jgi-psf.org/WebDB/CAPGROUP.FORM_RUN_SCAN_IN_MB400.show)
        **Reruns**

    b.  From the drop down menu, select a reason for rerun

    c.  Scan the plate barcode(s) into the **Rerun Plates** field

    d.  Click on **Submit**, and return the plate(s) to the Rm121 sample
        freezer to be re-assigned

**6. [Restarting the Instrument & Rebooting the Computer]{.underline}**

> In the event of an instrument or software issue, the following steps
> will restart the instrument and reboot the computer and software:

1.  While the instrument is stopped, close as many of the open software
    windows on the computer desktop as possible (some windows many not
    close if there is a software issue).

2.  Turn the instrument's power switch, located on the bottom right
    side, to the off position (a quick venting of the instrument's
    pressure system may be heard).

3.  Completely shut down the instrument's computer and power it off.

4.  Turn the instrument's power switch to the on position, wait \~30
    seconds for the system to initialize itself.

5.  Once the instrument is initialized, turn the computer on and allow
    it to boot-up:

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a.  While booting-up, the Notice to Users security screen will display,
    click **OK** and the computer will automatically log itself into the
    network (if the login is not automatic, refer to the User ID and
    Password posted on the top of the monitor to manually login).

b.  Once Microsoft Windows has started, the MegaBACE 4500 Host Scan
    Controller program will automatically start, allow it \~30 seconds
    to initialize.

    1.  Open the Instrument Control Manager program; its start icon is
        located in the MegaBACE folder on the computer's desktop.

**7. [Leaving the Instrument Idle & Storing the
Capillaries]{.underline}**

> Leaving the instrument idle means that the instrument power is on, but
> it is not running samples. If the instrument is left idle for 1 -- 7
> days, the capillaries need to be stored wet to prevent them from
> drying out. Leaving the instrument idle for more than 7 days requires
> the capillaries to be flushed and dried by MegaBACE instrumentation.
> The following steps will store the capillaries for a specified period:

1.  Materials Required:

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a.  Water tank with 120ml of Milli-Q water.

b.  Six clear 2mL micro-centrifuge tubes containing 1.75ml of Milli-Q
    water apiece.

    1.  In the **Instrument Control** window, select the **Sleep
        Parameters** tab; type the length of time the instrument will be
        idle in the Sleep Time field (1-168 hours).

    2.  In the **Instrument Control** window, with the **Store
        Capillaries** protocol selected, click on **START**. Follow the
        instructions on the instrument's display window to load the
        water tank into the left (cathode) side of each instrument
        followed by the 6 water tubes into the right (anode) side of the
        instrument.

> ***Important!** After the entered sleeping time has elapsed, it
> requires 3 full hours for the instrument to warm back up before it may
> be run again.*

**8. [Troubleshooting the MegaBACE 4500 DNA Analysis
System]{.underline}**

> Refer to the **MegaBACE 4000 DNA Analysis System Users Troubleshooting
> Guide** as a reference for troubleshooting various issues that may be
> encountered while operating the MegaBACE 4500 instrument.

# Reagent/Stock Preparation

**[1.5X Running Buffer]{.underline}**

1.  Add **300ml** MegaBACE 10X Running Buffer to a 2-L bottle.

2.  Add **1.7L** of Milli-Q water to the 2-L bottle.

3.  Mix well by inverting bottle.

4.  Label bottle: *lot number, date prepared, and initials*.

5.  Store in 4°C deli.

**[Preparing the MegaBACE 4500 2.6% V2E Matrix (LPA)]{.underline}**

1.  Remove the matrix tubes from their heat-sealed tray that they were
    shipped in and transfer them into the gray tube-blocks.

> ***NOTE:** Save the tray and lid. Take them to the washroom (Rm 143)
> where they will be washed and sent back to GE Healthcare for re-use.*

2.  Centrifuge the matrix in the blocks for **2 minutes at 3,000rpm**.

> ![](media/image1.png){width="0.375in"
> height="0.3020833333333333in"}***Important!** After centrifuging the
> matrix tubes, check for any empty tubes or tubes that still have
> bubbles in them. If empty tubes are found, set them aside and label
> them with the LPA lot# and date found. If bubbles are still present in
> any of the tubes, re-centrifuge them for 2 minutes at 3,000rpm.*

3.  After centrifuging the matrix tubes, check for any empty or low
    level tubes. If any are found write the lot number and date found on
    the side of the tube, put them aside and notify MegaBACE
    administration.

4.  Seal tubes with a clear plate seal and label: *lot number, date
    prepared, and initials*.

5.  Store in 4°C deli.

> ***NOTE:** Make sure to place the freshly made tubes in the back of
> the deli and bring the older tubes to the front.*

**[Preparing the MegaBACE 384-well Nunc Buffer Plates]{.underline}**

Refer to the **Multidrop/Twister Operation Protocol for MegaBACE 4000
384-well Buffer Plates**.

**[Preparing the 1.75ml 1.5X Buffer and 1.6ml Water Tubes used MegaBACE
instruments]{.underline}**

Refer to the **Tecan Miniprep 60 Operation Protocol for MegaBACE Water &
Buffer Tubes**.

# 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

Figure 1. Buffer tank loaded onto the left (cathode) side of the
MegaBACE 4500 instrument.

![](media/image2.jpeg){width="3.986111111111111in" height="3.0in"}

Figure 2. Six matrix tubes loaded onto the right (anode) side of the
MegaBACE 4500 instrument.

![](media/image3.jpeg){width="4.0in" height="3.0in"}

Figure 3. Water tank loaded onto the left (cathode) side of the MegaBACE
4500 instrument.

![](media/image4.jpeg){width="3.986111111111111in" height="3.0in"}

Figure 4. Sample plate with adapter loaded onto the left (cathode) side
of the MegaBACE 4500 instrument.

![](media/image5.jpeg){width="3.986111111111111in" height="3.0in"}

# Appendix B

**[MB4500 Loading Overview]{.underline}**

**1. [Necessary Loading Reagents & Supplies:]{.underline}**

> 1.1 [1.5X running buffer]{.underline}

a.  Diluted from 10X buffer stock -- 300ml of 10X buffer & 1700ml of
    Super-Q water mixed in a 2-L bottle

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2.  [Buffer Tank]{.underline} -- 120ml of 1.5X run buffer dispensed by
    Watson-Marlow pump

3.  [Water Tank]{.underline} -- 120ml of Super-Q water dispensed by
    Watson-Marlow pump

4.  [2.6% V2E Matrix (LPA]{.underline})

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a.  Kits are located on the bottom left shelf of the deli in Rm121, the
    trays are individually sealed in foil bags

b.  Transfer tubes from trays to gray bases marked with orange tape,
    spin them down at 3000rpm for 2min.

    2.  [1.5X run buffer plate]{.underline}

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a.  NUNC deep well plate filled with 85ul of 1.5X run buffer

b.  Prepare on Multidrop/Twister

    i.  In Configure Layout of Assist program change Plate Type to *384
        NUNC Plate*

    ii. Use *MB4500_1.5X_buffer_plates_85ul* program settings

    iii. Spin plates at 1000rpm for 1min

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    2.  [1.5X run buffer tubes]{.underline}

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    a.  2ml Fisher Microcentrifuge tubes filled with 1.75ml of 1.5X run
        buffer, use gray bases marked with blue tape to put the tubes
        in.

    b.  Prepare on Tecan Miniprep 60

        i.  Use *MB4500_1.75ml_Buffer_Tubes* program settings

        ii. Spin tubes at 1000rpm for 1min

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    3.  [MB Sample Plate]{.underline}

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a.  Injection Parameters: 2kV for 36sec

b.  Run Parameters: 5.9kv for 215min

**2. [Loading Scheme:]{.underline}**

> ***NOTE:** A single sequencing run on the MB4500s consists of loading
> sets of 7 instruments in 3 parts.*
>
> *Part 1: 7 instruments are started in a set according to Sections 2.1
> through 2.4.b.i of the MB4500 Users SOP.*
>
> ***NOTE:** Wait for all instruments in the set to finish their
> previous run prior to starting a new one.*
>
> *Part 2: Sections 2.4.b.ii & iii of the SOP are then performed on a
> subset of 4 & 3 instruments respectively.*
>
> *Part 3: Section 2.4.b.iv of the SOP is performed individually on each
> instrument of the subset.*
>
> *Finally, Parts 2 & 3 are repeated on the 2^nd^ subset of 3
> instruments.*
>
> *This is a descriptive outline of the MB4500 loading scheme:*

1.  Set out 120ml water reservoirs, 120ml 1.5X buffer reservoirs, and
    pre-spun V2E LPA for each of the 7 instruments in the Set.

2.  Start the **Matrix Fill and Prerun** protocol on each of the 7
    instruments:

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a.  As prompted by the instrument display, load the 120ml 1.5X buffer
    tanks into the cathode of each instrument.

b.  As prompted by the instrument display, load 6 tubes of V2E LPA into
    the anode of each instrument.

    1.  Prepare the DNA samples assigned to the Set according to the
        MB4500 Users SOP, and scan them into their corresponding
        instrument.

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a.  Sample Injection Parameters are 2kV for 36sec.

b.  Sample Run Parameters are 5.9kV for 215min.

    1.  Set out 85ul 1.5X NUNC run buffer plates and 1.75ml 1.5X run
        buffer tubes for each of the 7 instruments in the Set. Start the
        **Inject Samples and Run** protocol on each of the 7
        instruments:

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a.  As prompted by the instrument display, load the 120ml water tanks
    into the cathode of each instrument.

b.  For the first 4 instrument in the set:

    i.  Click **Enter** on the **Confirm to Continue** window on the
        first instrument of the set and as prompted load the assigned
        sample plate on its adapter into the cathode; repeat this for
        the remaining 2 instruments in the subset. ***Do not click Enter
        on all the instruments at once; this step must be done
        individually on each one.***

    ii. Once sample injection has completed on the first instrument in
        the subset of 3, click **Continue** on the **Confirm to
        Continue** window and as prompted load the 85ul 1.5X run buffer
        plate and 1.75ml 1.5X run buffer tubes into the cathode and
        anode respectively; repeat this for the remaining 2 instruments
        in the subset. ***Do not click Enter on all the instruments at
        once; this step must be done individually on each one.***

c.  After fully completing steps 2.4.b.i & .ii on the first 4
    instruments in the set, repeat these steps on the remaining 3
    instruments of the 2^nd^ subset.

# Appendix C

**[PROCEDURAL CHANGES]{.underline}**

**1/29/04 Stopping Blank Plates** -- Do not stop any more blank plates
(no labeled DNA sample present), even if the plate will post with a very
high failure rate. The reason for this policy change is that stopping
blank plates skews overall performance results, making readlengths and
pass rates appear higher. By letting these blank plates run and post, a
more accurate representation of performance is gained and allows for
better troubleshooting of the process line for areas that require
improvement. 

When a run that appears blank is encountered, continue the
troubleshooting steps of checking the current monitor, HSC, and zooming
in on the run electropherogram to see if the laser has cut off or if low
intensity sequencing peaks may be seen. Additionally, continue to
document these blank runs by taking screen shots of the run image and
notifying the MegaBACE Maintenance Technician or Lead Operator; make
note of it in your daily run log email as well.

**4/6/2004 Lab Hygiene: Buffer Spills --** Keep a damp towel handy while
loading instruments or doing prep work in order to wipe up buffer spills
as they occur; remember that wet buffer is much easier to clean-up than
allowing it to dry which requires scrubbing.

**5/17/2004 Saving HSC & HT Communication Data --** Save the Host Scan
Controller (HSC) and Hyper Terminal (HT) communication data from
instruments that experience the following hardware/software issues:

1.  HSC crashes (i.e. runs not starting/stopping, bad instrument status
    51 & 52, and general loss of communication errors).

2.  INTC/Board errors (i.e. CMON, TMPR, BEAM, ADAQ, SCAN, and EPHV board
    errors).

3.  Any unusual/uncommon errors (i.e. instrument not going to sleep/HSC
    still acquiring data errors).

HSC communication data may be saved on any instrument when one of the
above errors occurs. However, HT communication data may only be saved on
those instruments with the program installed, which are MB115, 116,
122 - 135, & RNDMB1. Refer to the **MegaBACE 4000 Comprehensive Users
Protocol** for instructions on saving HSC and HT communication data.

**7/12/2004 Adhering to Run Schedules --** Adhere to the posted MB run
sets and loading times as noted in the daily Run Schedule, unless
instructed otherwise by MegaBACE Administration or Instrumentation.
Furthermore, completely load one set before moving on to the next, do
not overlap sets (i.e. start the Matrix Fill protocol on one set while
still performing the Inject Samples and Run protocol on the previous
set). There are several reasons for the posted Run Schedule with set
start times, instruments split up into loading sets, and breaks between
some of the sets. The foremost of these are:

1.  Operator health and safety.

2.  Instrument well-being and prevention of catastrophic
    crashes/malfunctions.

3.  Prevention of operator error. Errors easily occur while rushing to
    load too many instruments at once or while loading overlapping sets,
    which can lead to harmful situations for both the operator and
    instrument.

4.  The ability to predict when instruments will be down, which is vital
    to the planning of instrument service and assigning priority
    samples.

5.  Minimization of variation. Introducing variation to any
    scientific/manufacturing process may lead to severe problems and be
    detrimental to performance; especially if the introduced variations
    are not properly noted and tracked.