MegaBACE 4000 DNA Analysis System

**Users Protocol**

> Version Number: 2.4
>
> Production Start Date: 12/05/03
>
> Version 2.4 Date: 08/18/04
>
> Author: Chris Daum
>
> Reviewed/Revised by: Nahal Lalefar

# Summary

This protocol outlines the operation of the MegaBACE 4000 DNA Analysis
System for high throughput capillary electrophoresis of DNA samples.

# Materials & Reagents

+----------------------------+-------------------------+--------------+
| **[Materials/Reagent       | *                       | **[Stock     |
| s/Equipment]{.underline}** | *[Vendor]{.underline}** | Number]{.    |
|                            |                         | underline}** |
| *                          | Axygen/ISC BioExpress   |              |
| [Disposables]{.underline}* |                         | T-3019-BCS   |
|                            | Edge Biosystems         |              |
| 384-well PCR Microplate,   |                         | 48461        |
| Blue                       | GE Healthcare\*         |              |
|                            |                         | 415002       |
| Clear Adhesive Plate       | GE Healthcare\*         |              |
| Sealers                    |                         | US79686      |
|                            | Millipore Milli-Q       |              |
| *[Reagents]{.underline}*   | System                  | \-           |
|                            |                         |              |
| MegaBACE 4000 Long Read    | GE Healthcare\*         | \-           |
| Matrix (LPA)               |                         |              |
|                            | Watson Marlow           | \-           |
| MegaBACE 10X Running       |                         |              |
| Buffer                     | VWR Scientific          | \-           |
|                            |                         |              |
| Milli-Q water              | VWR Scientific          | \-           |
|                            |                         |              |
| *[Equipment]{.underline}*  | Eppendorf               | \-           |
|                            |                         |              |
| MegaBACE 4000 Sequencer    |                         |              |
|                            |                         |              |
| Watson Marlow Pump         |                         |              |
|                            |                         |              |
| Heat Block                 |                         |              |
|                            |                         |              |
| Vortex                     |                         |              |
|                            |                         |              |
| Centrifuge 5810            |                         |              |
+----------------------------+-------------------------+--------------+

\* GE Healthcare, formerly Amersham Biosciences

# 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.

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 water tanks
    with **120ml** of Milli-Q water and buffer tanks with **120ml** of
    1X running buffer.

> ***Important!** Run 240ml additional Milli-Q water through the pump
> after running 1X 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.*

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 1X running buffer per instrument
    in the set.

b.  Six pre-spun (**2 minutes at 3,000rpm**) matrix tubes 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, with the **Matrix Fill and
    Prerun** protocol selected, click on **START** (perform this on each
    instrument in the set).

b.  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*.

> ![](media/image1.png){width="0.375in"
> height="0.3020833333333333in"}***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**.

    ii. Vortex the sample plates on the Vortexer for **2 minutes on
        setting 7**; loading no more than 12 plates on a Vortexer at a
        time.

    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, 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: 1.6 KV, Run Voltage:
         5.25 KV, Sample Injection Time: 36 sec, Run Time: 250 min.*

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    1.  Performing the **Inject Samples and Run** Protocol for a set of
        instruments:

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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 blue buffer plate containing 25μl
     of 1X running buffer per well.

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

b.  In the **Instrument Control** window, with the **Inject Samples and
    Run** protocol selected, click on **START**. After the **Select a
    Plate** window appears, scan in the barcode on the assigned sample
    plate (perform this on each instrument in the set).

> ***Important!** It is crucial to sample performance that the following
> steps are completed as soon as the instrument prompts that it is
> ready. Otherwise, a timeout error will occur and it may affect the
> quality of the sequencing run.*

i.  Follow the instructions on the instrument displays to load the water
    tank into the left 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*.

ii. Once the instruments finish their automated tip rinsing, the
    **Confirm to Continue** window appears, click on **ENTER** to
    continue.

iii. Follow the instructions on the instrument displays to load the
     assigned sample plate on the adapter into the left side of each
     instrument in the set; 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 while it is loaded. Remaining pieces of seal may
> harm the instrument if loaded.*

iv. After the instrument injects the samples, follow the instructions on
    the instrument display window to load the blue buffer plate on the
    adapter into the left side of each instrument in the set.

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

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
    matrix and buffer after the current run has finished.

> ***Important!** After the entered sleeping time has elapsed, it
> requires 2 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. The lack of an image after 40
        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, checking for regions of no current,
        abnormally high/low current, or erratic current that constantly
        changes values:

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

        ii. Groups of zeros or regions of abnormally high/low current
            may indicate more severe problems; notify MegaBACE
            instrumentation or administration immediately.

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 to
    <mb4000@cuba.jgi-psf.org>.

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 to <mb4000@cuba.jgi-psf.org>. 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
    (<http://venonat.jgi-psf.org/psfs/pfs.home>), 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
        (<http://venonat.jgi-psf.org/psfs/pfs.home>), 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 - Dump and shake out each buffer
        tank into the large funnel above the carboy in 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. When the carboy containing disposed of LPA/Buffer reaches ¾ of
         the way full, fill it the rest of the way with hot tap water.

    iv. Open the spigot at the bottom of the carboy so that the waste
        drains into the sink.

    v.  Turn on the hot tap water all the way so that the waste becomes
        as dilute as possible as it goes down the drain.

    vi. When the carboy is empty close the spigot and continue to let
        the water run for at least 5 minutes to flush the sink's drain.

**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.

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**).

**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 4000 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 2-ml tubes, each containing 1.6ml of Milli-Q water.

    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 2 full hours for the instrument to warm back up before it may
> be run again.*

**8. [Rinsing the Capillary Tips]{.underline}**

> Use the Rinse Tips protocol to clean capillary tips, minimize
> contamination of the electrodes, and to prevent clogging of the holes
> in the circuit board. It is important to run this protocol after
> storing the instrument for long periods or leaving it idle in
> matrix/buffer for long periods.

1.  Materials Required:

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

b.  Six 2-ml tubes, each containing 1.6ml of Milli-Q water.

    1.  In the **Instrument Control** window, with the **Rinse Tips**
        protocol selected, click on **START**. Follow the instructions
        on the instrument's display windows 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.

**9. [Troubleshooting the MegaBACE 4000 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 4000 instrument.

# Reagent/Stock Preparation

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

1.  Add **200ml** MegaBACE 10X Running Buffer to a 2-L bottle.

2.  Add **1.8L** 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 4C deli.

**[Preparing the MegaBACE 4000 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 4C 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 Buffer Plates]{.underline}**

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

**[Preparing the 1.6ml Water Tubes for storing & rinsing tips on the
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 4000 instrument.

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

Figure 2. Six matrix tubes loaded onto the right (anode) side of the
MegaBACE 4000 instrument.

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

Figure 3. Water tank loaded onto the left (cathode) side of the MegaBACE
4000 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 4000 instrument.

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

# Appendix B

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

**5/27/04** -- Alla Lapidus and Tijana Glavina performed an audit
between 11:00AM -- 12:35PM. The operator was Albert Linkowski. Changes
from audit incorporated **8/04/04**.

**[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.