### **Post-Sequencing BET Cleanup Protocol**

### **(384-well plates for MB4000 and ABI3730)**

Automated Process (Biomek FX)

> Version Number: 3.3
>
> Production Start Date: 12/15/03
>
> Version 3.3 Date: 7/21/05
>
> Authors: Christine Lansang, Nancy Hammon, Steven Wilson, Catherine
> Gordon, Arshi Khan, Amber Nivens
>
> Reviewed/Revised by: Cailyn Spurrell

# Summary

The purpose of this procedure is to automate the BET cleanup protocol
using Biomek FX robots. This process is the post-sequencing reaction
cleanup procedure for the BigDye and ET-Terminator cycle sequencing
reactions of RCA and Fosmid production plates using magnetic beads, 70%
ethanol, and TEG reagents. The program uses P30 pipette tips, custom
magnets, four plate stackers, a series of deck positions and a wash
station. The final product is a plate with clean resuspended, dye
labeled DNA fragments to run on the MegaBACE 4500 or the ABI 3730xl
sequencer

s\.

# Materials & Reagents

+------------------------------+---------------------+-----------------+
| **[Materials/Reage           | **[Ve               | **[Stock        |
| nts/Equipment]{.underline}** | ndor]{.underline}** | Number          |
|                              |                     | ]{.underline}** |
| *[Disposables]{.underline}*  | Axygen/ISC          |                 |
|                              | BioExpress          | T-3059-1CS      |
| 384-well PCR Microplate,     |                     |                 |
| Clear                        | Applied Biosystems  | 4343814         |
|                              |                     |                 |
| 384-well PCR Microplate,     | Corning/VWR         | 430290          |
| Clear                        |                     |                 |
|                              | Falcon/VWR          | 357550          |
| 50-ml Centrifuge Tube        |                     |                 |
|                              | Falcon/VWR          | 357525          |
| 50-ml Serological Pipet      |                     |                 |
|                              | VWR                 | BK719222        |
| 25-ml Serological Pipet      |                     |                 |
|                              | Matrix              | 7151            |
| Biomek AP 384 Pipette tips   |                     |                 |
|                              | Velocity 11         | 06644-001       |
| 250-μl Matrix Pipette tips   |                     |                 |
|                              | Cole Parmer         | 06485-36        |
| PlateLoc Pierceable Seal     | Instrument Co.      |                 |
|                              |                     | 96400-24        |
| MasterFlex Pharmed Tubing    | Cole Parmer         |                 |
|                              | Instrument Co.      | EW-95603-44     |
| MasterFlex Silicone Tubing   |                     |                 |
|                              | Cole Parmer         | EW-95603-48     |
| Ismatec 3-stop tubing        | Instrument Co.      |                 |
| (2.29mm ID)                  |                     | 48461           |
|                              | Cole Parmer         |                 |
| Ismatec 3-stop tubing        | Instrument Co.      | \-              |
| (2.79mm ID)                  |                     |                 |
|                              | Edge BioSystems     | 110175-KG       |
| Clear Adhesive Plate Sealers |                     |                 |
|                              | \-                  | 44152105050350  |
| Tri-Flow Industrial          |                     |                 |
| lubricant                    | Sigma-Aldrich       | 6810-13289      |
|                              |                     |                 |
| *[Reagents]{.underline}*     | Agencourt           | \-              |
|                              |                     |                 |
| Tetra-Ethylene Glycol        | AAPER Alcohol       | **[Stock        |
|                              | (LBNL)              | Number          |
| Seradyn Magnetic             |                     | ]{.underline}** |
| Carboxylate-Modified Beads   | Millipore Milli-Q   |                 |
|                              | System              | 719222          |
| 100% Ethanol (200 Proof)     |                     |                 |
|                              | **[Ve               | \-              |
| Milli-Q Water                | ndor]{.underline}** |                 |
|                              |                     | \-              |
| **[Materials/Reage           | Beckman Coulter/VWR |                 |
| nts/Equipment]{.underline}** |                     | \-              |
|                              | Clay Adams          |                 |
| *[Equipment]{.underline}*    |                     | \-              |
|                              | Promega             |                 |
| Biomek FX                    |                     | \-              |
|                              | JGI                 |                 |
| Vari-Mixer                   |                     | \-              |
|                              | Eppendorf           |                 |
| MagneSil Magnetic Separation |                     | \-              |
| Unit                         | Spectramax          |                 |
|                              |                     | \-              |
| 384-well High Field Plate    | Velocity 11         |                 |
| Magnet                       |                     |                 |
|                              | Eppendorf           |                 |
| Centrifuge 5810              |                     |                 |
|                              | Matrix              |                 |
| Spectramax 384 Plus          |                     |                 |
| Spectrophotometer            |                     |                 |
|                              |                     |                 |
| PlateLoc Thermal Plate       |                     |                 |
| Sealer                       |                     |                 |
|                              |                     |                 |
| Easypet                      |                     |                 |
|                              |                     |                 |
| 8-channel Pipette (125μl)    |                     |                 |
+------------------------------+---------------------+-----------------+

# 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.

# Calibration Check

**[Biomek FX]{.underline}**

Tips should be changed every 2-3 days or when they appear dirty. A tip
QC should be performed when new tips are installed. Additionally, the
dye QC test requires a plate weight reading. Refer to the **QC Protocol
for the Biomek FX Liquid Handler**.

# Procedure 

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

**1. [Preparing samples for Biomek FX cleanup procedure]{.underline}**

1.  Remove MegaBACE 4500 and/or ABI 3730 plates from the TEG Backlog
    deli (the 4^o^C fridge \# 407 in Rm141).

> ***NOTE 1:** Twenty plates can be loaded per Biomek FX per run.*
>
> ***NOTE 2:** ABI plates need to be loaded with other ABI plates and
> MegaBACE plates need to be loaded with other MegaBACE plates. Fosmid
> plates (although they are ABI plates) need to be loaded with other
> Fosmid plates. However, if running 20 ABI plates, you can have 10
> consecutive Fosmid plates and 10 consecutive ABI plates. Do not mix
> Fosmids and ABI plates within a group of 10 plates.*

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

3.  Remove the seals from the plates and throw the seals away in the
    autoclave waste. Create stacks of no more than 20 plates per Biomek
    FX.

> ***NOTE 1**: Do not shake plates and dislodge sample.*
>
> ***NOTE 2**: If source plates are not going to be loaded onto the
> Biomeks within 10 minutes, place a clear seal (labeled with correct
> Biomek FX #) on the top plate in the stack and store plates in the TEG
> Backlog deli.*

**2. [Creating barcodes for destination plates]{.underline}**

1.  For MegaBACE 4500 plates, go to the Venonat database
    (<http://venonat.jgi-psf.org/psf/PSF.home>)  click on **RCA DB** 
    **Step 5 Scan (MegaBACE) Plates for TEG:**

    a.  Select the correct information from the pull down menus:
        *Operator, TEG Instrument, Bead Lot, TEG Lot, Ethanol Lot,
        Clamps Used?, Posting Type.*

    b.  Use the barcode scanner to scan in the stack of source plates.

    c.  Click on **SUBMIT** & Print the conformation page.

    d.  Click on **Print Barcodes**.

    e.  Select **RCA (R140)** and print.

    f.  Affix the barcodes to new Axygen plates (on the numbered and
        indented side of the plate), creating destination plate stacks
        that match the order of the source plate stacks.

> ***NOTE 1:** Wear gloves when affixing barcodes to destination
> plates.*
>
> ***NOTE 2:** This step may be performed after the Biomek FX program
> has started running.*
>
> ***NOTE 3:** Mark the barcodes for priority plates with a red line.
> Mark plates that come with a plate request with "PR". Mark Microbe QC
> plates with "MQC". Mark any unpaired plate with a blue "S" & when the
> run is complete place in the bin marked unpaired in the megabase
> backlog freezer in 140.*
>
> ***NOTE 4:** After labeling the destination plates, assure that the
> order of source plates matches the order of destination plates found
> in each stack. This can be done by double checking the plates with the
> printed conformation page, placing the source stack and destination
> stack side by side and visually scanning over the plates, double
> checking that the first and last plate of each source and destination
> stack are matches, or (if you've already loaded the source plates onto
> the Biomek and have started the run), double check that at least one
> of the first destination plates to be loaded onto the Biomek matches
> at least one of the first source plates already being processed on the
> Biomek.*
>
> ***NOTE 5***: *If destination plates are not going to be loaded onto
> the Biomeks within 10 minutes, place a clear seal (labeled with
> correct FX #) on the top plate of each stack.*

2.  For ABI 3730 plates, go to the Venonat database
    (<http://venonat.jgi-psf.org/psf/PSF.home>)  click on **RCA DB** 
    **Step 5 Scan (ABI) Plates for TEG (Associations)**:

    a.  Repeat steps **2.1.a** -- **2.1.e.**

    b.  If using the CyBio Print & Apply to label plates refer to the
        addendum at the end of this protocol and continue with step 3
        *Biomek FX Setup* of this protocol, otherwise continue with step
        2.2.c.

> ***NOTE**: Label one ABI batch per shift using the CyBio Print &
> Apply.*

c.  Affix the barcodes to the long side of new ABI plates (the side
    without an ABI barcode), creating destination plate stacks that
    match the order of the source plate stacks.

> ***NOTE 1:** Wear gloves when affixing barcodes to destination
> plates.*
>
> ***NOTE 2:** This step may be performed after the Biomek FX program
> has started running.*
>
> ***NOTE 3**: Mark Fosmid barcodes with a regular green line & priority
> barcodes with a red line. Mark plates that come with a plate request
> with "PR" & mark Microbe QC plates with "MQC". If a pair is split for
> some reason mark the ABI barcode with a blue "S" and place in the ABI
> unpaired bin in the ABI backlog deli.*
>
> ***NOTE 4**: After labeling the destination plates, assure that the
> order of source plates matches the order of destination plates found
> in each stack. This can be done by double checking the plates with the
> printed conformation page, placing the source stack and destination
> stack side by side and visually scanning over the plates, double
> checking that the first and last plate of each source and destination
> stack are matches, or (if you've already loaded the source plates onto
> the Biomek and have started the run), double checking that at least
> one of the first destination plates to be loaded onto the Biomek
> matches at least one of the first source plates already being
> processed on the Biomek.*
>
> ***NOTE 5***: *If plates are not going to be loaded onto the Biomeks
> within 10 minutes, then place a clear seal (labeled with correct FX #)
> on the top plate of each stack.*

**3. [Biomek FX Setup]{.underline}**

1.  Check to make sure that the main water valve of the Biomek FX is in
    the **ON** position.

2.  Set up the reagent reservoirs:

    a.  Attach the 500-ml reagent bottles (BET, ethanol, Milli-Q water)
        to the orange caps connected to the tubing.

> ***NOTE 1:** Total of 12 bottles needed for all 4 Biomek FX
> instruments.*
>
> ***NOTE 2:** Use freshly made BET reagent for the first shift of the
> day, and recycled BET reagent for the 2^nd^ & 3^rd^ shift of the day.
> Refer to **Reagent/Stock Preparation** to see how to prepare BET
> solutions.*
>
> ***Checkpoint:** Check to make sure that the waste bottle is in
> place.*

b.  Snap the Ismatec Return and Supply applicators down on the Ismatec
    pump.

> ***Checkpoint:** Check to see if the Ismatec tubing needs to be moved
> or changed before snapping down the applicators. If it is a Monday,
> remember to move and date the MasterFlex tubing or enter a work
> request if it needs to be changed.*
>
> ***NOTE:** Refer to the **Biomek Instrument Maintenance** section for
> moving or changing the Ismatec tubing or moving the MasterFlex
> tubing.*

c.  Turn the Ismatec pump on to the forward direction.

> ***Checkpoint:** Make sure the Ismatec pump speed is set at 25, the
> reagent reservoirs are in the correct positions, and that the reagents
> are flowing into the reservoirs.*

3.  If this is the first run of the day, initialize the Biomek carousel
    by using the remote keypad (Stacker Carousel Pendant) connected to
    the carousel.

    a.  Press the **Remote Local** button. The display on the keypad
        should read "Keypad Control Only."

    b.  Press the **Initialize** button. A message will appear on the
        keypad reading "Press initialize key to initialize or press
        another key to cancel." Press the **Initialize** button again.

    c.  The carousel will slowly rotate 360°. When finished rotating
        press the **Remote** **Local** button to return the stacker
        control to the Biomek FX. The display should read "Press the
        Remote Local Key."

4.  Load the desired number of source plates into Stacker A, starting at
    the bottom of the hotel with labels facing inward (A1 position out).

> ***NOTE 1:** Each stacker may hold a maximum of 20 plates.*
>
> ***NOTE 2:** Manual control of the stacker for loading can be
> accomplished by using the Stacker Carousel Pendant connected to the
> carousel. The display on the keypad should read "Keypad Control Only"
> for manual control. If the display reads "Press the Remote Local Key,"
> press the **Remote Local** button on the keypad to bring the display
> to read "Keypad Control Only." Once in manual control, use the
> **Rotate Right** and **Rotate Left** buttons on the keypad to rotate
> the carousel.*

5.  Load the set of destination plates into Stacker C.

> ***NOTE:** This step does not need to be done right away. It can be
> done within the first 45 minutes of starting the run.*

6.  Open the **Biomek FX** program on the desktop.

7.  Open the appropriate program file:

```{=html}
<!-- -->
```
a.  Click on **File**  **Open**  **Methods**  **TEG_FLAT+MAGS+BCR\_
    SW_0909.04.bmt**

b.  Click on **Open**.

    1.  If this is the first run of the day, make sure that the Biomek
        power is turned on and then home all axes by going to **Tools
        ** selecting **Manual Control ** select **Home All Axes**.

> ***NOTE 1**: Make sure the grippers on POD 2 are up before you home
> the axes.*
>
> ***NOTE 2:** A pop-up box with a series of conditions will appear,
> make sure all conditions are met before hitting OK.*
>
> ***NOTE 3**: Homing all axes takes approximately 30 seconds. When the
> pods are no longer moving you can exit the **Home All Axes** program*.

2.  After checking the levels of the reagent reservoirs, click on the
    **RUN** (green arrow) button.

> ***NOTE:** The display on the Stacker Carousel Pendant should read
> "Press the Remote Local Key" for Biomek FX control. If the display
> reads "Keypad Control Only," press the **Remote Local** button on the
> keypad to return the display to "Press the Remote Local Key."*

3.  Upon prompt, enter the number of **source plates** to be run and
    select OK.

4.  Another prompt will ask for the number of **MegaBACE** plates.

```{=html}
<!-- -->
```
a.  If running *MegaBACE* plates:

    i.  Enter the number of MegaBACE source plates when prompted and
        select **OK**.

> ***NOTE:** ABI and MegaBACE plates cannot be mixed. If the number of
> MegaBACE plates is greater than 0, all plates will be run as MegaBACE
> plates.*

b.  If running *ABI* plates:

```{=html}
<!-- -->
```
i.  Leave the default **0** for the number of MegaBACE plates and select
    OK.

```{=html}
<!-- -->
```
c.  Look at the Biomek FX deck layout and make sure it matches the setup
    on the computer diagram.

> ***Checkpoint:** Make sure that the reservoirs are full, the clamp
> switch is on, the Ismatec pump is on and running, the FX controller
> device box switch is on automatic, and the water source switch is on.*

d.  When ready, click **OK**, and observe Biomek FX to make sure tips
    are aspirating and transferring liquid.

> ***NOTE 1:** The entire process takes approximately 60 minutes for
> every 10 plates loaded. When running 20 plates, the total run time is
> approximately 2 hrs for ABI plates and 2 hrs 15 min for MegaBACE
> plates*.
>
> ***NOTE 2:** The Biomek FX has a light curtain, which protects the
> deck area during operation. If this curtain is crossed during
> operation, the protocol will stop and prompt a light curtain error. To
> reset the light curtain, click on the "OK" button on the prompt
> window*.
>
> ***NOTE 3***: *Plate counts are recorded in an excel spreadsheet
> entitled "TEG Plate Count." At some point during the run enter in the
> correct number of ABI or MB plates processed for that batch. The "TEG
> Plate Count" spreadsheet can be found in OctopusProd SeqRCATEG
> Plate Count.*
>
> ***Checkpoint:** It is important to look for consistent levels of
> reagents at the respective stages. One of the most important steps to
> observe is the ethanol wash procedure. After adding and removing the
> ethanol from the plates, there should be no ethanol left over in the
> wells, only dry magnetic beads on the bottom of wells.*

1.  When the BET cleanup is complete, carefully unload plates from
    Stackers B and D as to not mix up the order.

> ***NOTE:** The source plates will be in stacker B and the destination
> plates will be in stacker D.*

2.  After each set of 10 plates, a **Barcode Verification and
    Submission** program window will open:

```{=html}
<!-- -->
```
a.  If the window is gray, then all barcodes were successfully scanned
    and no mix-ups occurred:

    i.  Fill in the requested information: *Operator's initials, and any
        comments*.

    ii. Click on **Proceed.**

    iii. Click on **Upload.**

b.  If the window is yellow, the barcode scanner did not scan one or
    more barcodes:

    i.  Carefully identify the plates that need to be scanned into the
        form (matching source and destination plates) and scan them in
        manually using the handheld scanner. Select the appropriate
        reason from the drop down menu of why the barcode was not
        scanned in on the biomek.

    ii. Repeat steps **3.13.a.i -- 3.13.a.ii**.

c.  If the window is red, then a mix-up occurred. *Notify a supervisor
    or instrumentation specialist immediately to determine how to
    proceed*. Normally, if the mix-up is simple in nature and can be
    fixed with 100% confidence, the operator can correct the mix-up by
    switching the barcodes on the destination plates. If the mix-up is
    complex or cannot be fixed with 100% confidence, then all of the
    plates involved must be failed. Check the **Do not submit** box next
    to the affected plates, and fill in the requested information upon
    submitting. These plates must also be scanned into the **Enter Fails
    or Comments** form in the **RCA DB** as failures. Print out a copy
    of these failed plates and give to Sequencing Chemistry. Mark on the
    sheet if these are ABI or MegaBACE plates. An [**Incident
    Report**](../../../../../../../../ProdSeq/Incident%20Reports) must
    also be written.

    1.  When the BET cleanup is complete and the barcodes verified:

```{=html}
<!-- -->
```
a.  Discard the source plates from Stacker B into the autoclave waste.

b.  Prepare and store the plates for sequencing:

> ***NOTE:** For passing on unpaired plates, label the JGI barcode with
> a blue "S" and place it in the appropriate refrigerator or freezer
> specified below within the "unpaired" bin.*

i.  For the *MegaBACE* plates:

    1.  Seal the plates with clear plate seals.

    2.  Store the plates (match FW and RV pairs) in the MegaBACE Backlog
        freezer (the -20^o^C freezer 201 in Rm140) in bins labeled with
        MB & the BET cleanup date.

> ***NOTE**: In some situations, if you don't have time to seal each
> plate, you can temporarily store plates in the TEG backlog deli. You
> should seal the top plate of each stack and keep plates there for no
> longer than 1-2 hours.*

ii. For the *ABI* plates:

    1.  Seal the plates with the PlateLoc foil sealer.

    2.  Visually check each plate to make sure all wells have sample.

    3.  Store the plates (match FW and RV pairs) in the ABI Backlog deli
        (the 4^o^C fridge #406 in Rm141) in bins labeled with ABI & the
        BET cleanup date.

    ```{=html}
    <!-- -->
    ```
    1.  The Biomek FX can now be loaded for another run.

**4. [Biomek FX Cleanup ]{.underline}**

If there are no more runs for the day, then the following steps should
be carried out:

1.  Switch the Ismatec pump to the "reverse" direction and prop the
    reservoirs at an angle so that their reagents are fully emptied.
    Stop the pump once lines are empty.

2.  Remove the orange caps from the reagent bottles.

    a.  On Monday-Thursday consolidate the remaining BET solution from
        all 4 biomeks into one bottle (labeled BET & current date) and
        store it in the 4° C TEG deli located in Rm 141.

> ***NOTE**: After consolidating the remaining BET solution, make sure
> to track the amount of solution in the BET Tracking spreadsheet. The
> "BET tracking" spreadsheet can be found in OctopusProd SeqRCATEG
> Plate Count.[**\\\\Octopus\\ProdSeq\\RCA\\TEG Plate
> Count.xls**](../../../../../../../../ProdSeq/RCA/TEG%20Plate%20Count.xls)*

b.  On Friday dump the remaining BET solution into the red Hazardous
    Waste Container.

> ***NOTE 1:** If the Hazadous waste container is empty, you will need
> to mark it with the date that liquid started being collected in the
> container.*
>
> ***NOTE 2:** Make sure to note the amount of BET solution that was
> thrown out under the Comments section in the BET tracking
> spreadsheet.*
>
> 4.3 Replace the BET bottle with the Milli-Q water bottle.
>
> 4.4 Release the water and ethanol pump white applicators and continue
> to pump the water in the forward direction to clean the BET reservoir
> with water.
>
> ***NOTE:** Pump in enough water to cover the bottom of the BET
> reservoir.*
>
> 4.5 Reverse the water flow from the BET reservoir and prop the
> reservoir at an angle to ensure the removal of any residual water.
> Stop the pump once lines are empty.
>
> 4.6 Remove the BET cap from the Milli-Q water bottle, dump the Milli-Q
> water bottle contents into the sink, and attach the 70% ethanol bottle
> to the BET cap.
>
> 4.7 Pump the ethanol in the forward direction to clean the BET
> reservoir.
>
> ***NOTE:** Pump in enough ethanol to cover the bottom of the BET
> reservoir.*
>
> 4.8 Wipe the BET, water, and ethanol reservoirs with damp paper
> towels.
>
> ***NOTE**: Make sure to wipe BET trough separately than the other
> troughs as to not contaminate the other troughs with residual beads.*
>
> 4.9 Reverse the ethanol flow from the BET reservoir and prop the
> reservoir at an angle to ensure the removal of any residual ethanol.
> Stop the pumps once the lines are empty.
>
> 4.10 Remove the BET cap from the ethanol bottle and release the BET
> Return and Supply white applicators.
>
> 4.11 Wipe all plate flatteners with 70% ethanol.
>
> 4.12 Pour the 70% Ethanol bottle contents into the red Hazardous Waste
> Container.
>
> ***NOTE:** If the waste container is empty, you will need to mark it
> with the date that liquid started being collected in the container.*
>
> 4.13 Remove the orange cap from the waste bottle and dispose of the
> waste into the designated red Hazardous Waste Container.

14. Place the waste bottle back on the Biomek.

> ***NOTE:** Replace the waste bottle with a new autoclaved 2L bottle
> every Friday.*
>
> 4.15 Place all bottles in the autoclave bin located next to the sink.
>
> 4.16 Close out the software window by selecting **File**  **Exit**.
> If prompted to save changes, click on **NO**.

# Reagent/Stock Preparation

NOTE: Wear a lab coat and gloves when preparing reagents. You MUST wear
goggles when preparing 70% Ethanol.

**[Washed Bead Reagent]{.underline}**

> ***NOTE:** Three bottles of beads, needed to make 16 tubes of washed
> bead reagent, should be taken out of the QC deli 3 days ahead of time
> and placed on the Vari-Mixer.*

1.  Add **15ml** of thoroughly mixed Seradyn beads to a 50-ml Falcon
    tube.

2.  Add **35ml** of Milli-Q water.

3.  Place the tube on the Promega magnet for 15 minutes or until the
    solution is almost clear.

4.  With the tube on the magnet, pour off the water.

5.  Repeat steps **2** -- **4** two more times.

> ***NOTE:** Rotate the tube 180^o^ after each wash to ensure that all
> of the beads get thoroughly washed.*

6.  Add **15ml** of Milli-Q water to the Falcon tube.

7.  Label tube: *Washed beads, Bead lot number, date, operator
    initials*.

8.  Place on a Vari-Mixer until ready for use.

**[Fresh BET Solution Preparation]{.underline} (prepared daily during
first shift)**

1.  The following reagents are required to process 240 Axygen 384-well
    plates (3 batches of 20 plates on each of the 4 Biomeks). In a 2-L
    bottle combine the following reagents:

> ***NOTE**: You will have approximately 349mls of BET solution left
> over at the end of the 3^rd^ batch.*

+------------------------------------------+---+---------------------------+
| > **Reagent**                            |   | **Volume (16X)**          |
+------------------------------------------+---+---------------------------+
| 100 % Ethanol (200 proof)                | > |                           |
|                                          |   |                           |
|                                          | 1 |                           |
|                                          | 0 |                           |
|                                          | 2 |                           |
|                                          | 4 |                           |
|                                          | > |                           |
|                                          |   |                           |
|                                          | m |                           |
|                                          | l |                           |
+------------------------------------------+---+---------------------------+
| > Milli-Q water                          | > |                           |
|                                          |   |                           |
|                                          | 1 |                           |
|                                          | 1 |                           |
|                                          | 2 |                           |
|                                          | > |                           |
|                                          |   |                           |
|                                          | m |                           |
|                                          | l |                           |
+------------------------------------------+---+---------------------------+
| > Tetra Ethylene Glycol                  | > |                           |
|                                          |   |                           |
|                                          | 1 |                           |
|                                          | 0 |                           |
|                                          | 2 |                           |
|                                          | . |                           |
|                                          | 4 |                           |
|                                          | > |                           |
|                                          |   |                           |
|                                          | m |                           |
|                                          | l |                           |
+------------------------------------------+---+---------------------------+
| > Washed Reagent Bead                    | > |                           |
|                                          |   |                           |
|                                          | 3 |                           |
|                                          | 2 |                           |
|                                          | > |                           |
|                                          |   |                           |
|                                          | m |                           |
|                                          | l |                           |
+------------------------------------------+---+---------------------------+

2.  Consult the *BET and 70% EtOH Solution Preparation* sheet located at
    the bottom of this section to determine how much solution should be
    prepared based on the number of plates that need to be processed.

3.  Dispense the appropriate volume of ethanol and water into the 2-L
    bottle.

4.  Add the appropriate amount of TEG. After dispensing TEG with a
    serological pipet, aspirate the TEG/water/ethanol mixture into the
    pipette to remove any residual TEG on the walls of the pipet.

5.  Add the washed bead reagent. Cap the bottle and mix by inverting and
    shaking the bottle.

> ***NOTE:** If reagents are not going to be used within the next 20
> minutes store the BET in the ABI Backlog deli in Rm141.*

**[Recycled BET Solution Preparation (prepared daily for 2^nd^ & 3^rd^
shift)]{.underline}**

1.  Obtain the previous day's consolidated BET solution from the TEG
    deli located in Rm 141.

2.  Consult the *BET and 70% EtOH Solution Preparation* sheet located at
    the bottom of this section to determine how much solution should be
    prepared based on the number of plates that need to be processed.

3.  Subtract the amount of leftover BET solution from the total amount
    of BET needed to determine the amount of fresh BET to prepare.

4.  Dispense the appropriate volume of ethanol and water into the 2-L
    bottle.

5.  Add the appropriate amount of TEG. After dispensing TEG with a
    serological pipet, aspirate the TEG/water/ethanol mixture into the
    pipette to remove any residual TEG on the walls of the pipet.

6.  Add the washed bead reagent.

7.  Add the consolidated BET solution from the previous day. Cap the
    bottle and mix by inverting and shaking the bottle.

> ***NOTE:** If reagents are not going to be used within the next 20
> minutes store the BET in the ABI Backlog deli in Rm141.*

**[70% Ethanol Wash Solution]{.underline} (prepared fresh daily)**

**[Consult the *BET and 70% EtOH Solution Preparation* sheet located in
the BET Cleanup bay to determine how much solution should be prepared
based on the number of plates that need to be processed. Add the
following reagents to a 2-L bottle (to process 240 Axygen 384-well
plates) and mix by inverting and shaking:]{.underline}**

  ------------------------------------------ ----------------------------
  **Reagent**                                **Volume (22X)**

  100 % Ethanol (200 proof)                  1540 ml

  Milli-Q water                              660 ml
  ------------------------------------------ ----------------------------

> ***NOTE 1**: EtOH bottles can be found and signed out of the Flammable
> Cabinet in Rm 141*.
>
> ***NOTE 2***: *If reagents are not going to be used within the next 20
> minutes store the 70% EtOH in the ABI Backlog deli in Rm141.*
>
> ***NOTE 3:*** *Graduated cylinders must be covered with foil when not
> in use. Funnels should be washed once per week. Graduated cylinders
> should be washed and autoclaved once per week.*

  ------------- ------------ --------------- ----------- --------- ----------- ---------- ------------ ---------- ------------ ------------ ------------ ------------ ------
                             **BET and EtOH                                                                                                                           
                             Solution                                                                                                                                 
                             Preparation**                                                                                                                            

                                                                                                                                                                      

                                                                                                                                                                      

  **BET         **Volume**   **2X**          **4X**      **5X**    **8X**      **10X**    **16X**      **20X**    **22X**      **24X**      **26X**      **28X**      
  Solution**                                                                                                                                                          

  Ethanol 200   64 ml        128             256         320       512         640        1024         1280       1408         1536         1164         1792         
  proof                                                                                                                                                               

  Milli-Q Water 7 ml         14              28          35        56          70         112          140        154          168          182          196          

  Tetra         6.4 ml       12.8            25.6        32        51.2        64         102.4        128        140.8        153.6        166.4        179.2        
  Ethylene                                                                                                                                                            
  Glycol                                                                                                                                                              

  Washed        2 ml         4               8           10        16          20         32           40         44           48           52           56           
  Seradyn beads                                                                                                                                                       

  **Total       **79.4**     **158.8**       **317.6**   **397**   **635.2**   **794**    **1270.4**   **1588**   **1746.8**   **1905.6**   **2064.4**   **2223.2**   
  Volume (ml)**                                                                                                                                                       

                                                                                                                                                                      

  **70% EtOH**  **Volume**                                                                                                                                            

  Ethanol 200   70 ml        140             280         350       560         700        1120         1400       1540         1680         1820         1960         
  proof                                                                                                                                                               

  Milli-Q water 30 ml        60              120         150       240         300        480          600        660          720          780          840          

  **Total       **100**      **200**         **400**     **500**   **800**     **1000**   **1600**     **2000**   **2200**     **2400**     **2600**     **2800**     
  Volume (ml)**                                                                                                                                                       

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

# Instrument Setup & Maintenance

**[Biomek FX]{.underline}**

**Moving the Ismatec Tubing:**

***NOTE:** When moving or changing the Ismatec tubing, make sure the
Biomek is not running.*

1.  Switch the Ismatec pump off.

2.  One at a time, release an Ismatec pump white applicator, dissociate
    it from its tubing, and move the white applicator over one notch.

3.  After all of the white Return and Supply applicators have been
    moved, sparingly spray the bottom of the tubing that is now under
    the white applicators with Tri Flow industrial lubricant.

> ***NOTE:** Using liberal amounts of lubrication spray can reduce
> tubing lifetime. Tubing particles can gather beneath clamp and tear
> tubing.*

4.  Switch the Ismatec pumps off.

5.  Re-attach the Ismatec pump white applicators and snap down onto the
    Ismatec pump rotating device.

> ***NOTE:** Make sure to place the applicators back in the same order.*

6.  Switch the Ismatec pump on to the forward direction.

7.  Mark that the tubing has been moved.

**Changing the Ismatec Tubing:**

1.  Switch the reagent Ismatec pump to the reverse direction and prop
    the reservoirs at an angle so that their reagents are fully emptied.

2.  Once all reservoirs and tubing are emptied, switch the Ismatec pump
    off.

3.  One at a time, release an Ismatec pump white applicator, dissociate
    it from its tubing, remove the old tubing and place it in the
    autoclave waste.

4.  Wipe tubing residue from each white clamp with a dry paper towel.
    This will help extend tubing lifetime.

5.  Replace with new tubing of the same color and re-attach the white
    applicator to the tubing.

> ***NOTE:** All supply lines should have purple and gray tubing; all
> return lines should have purple and white tubing.*

6.  After all of the tubing has been changed, sparingly spray the bottom
    of the tubing that is now under the white applicators with Tri Flow
    industrial lubricant.

7.  Switch the Ismatec pump off.

8.  Re-attach the Ismatec pump white applicators and snap down the
    applicators onto the Ismatec pump rotating device.

> ***NOTE:** Make sure to place the applicators back in the same order.*

9.  Switch the Ismatec pump on to the forward direction.

10. Place a piece of tape onto the Ismatec pump motor labeled with what
    day the tubing needs to be moved (one week later) and what day the
    tubing needs to be changed (2 weeks later).

**Moving and Changing the MasterFlex Tubing:**

***NOTE:** When moving or changing the MasterFlex tubing, make sure the
Biomek is not running.*

1.  At the beginning of every week, release the MasterFlex tubing
    clamps, move the MasterFlex tubing over one notch, and reclamp the
    MasterFlex tubing clamps. Mark the date it was moved on the tubing.

2.  There are four spaces that the MasterFlex tubing can be moved. When
    all spaces have been filled (approx. 1 month), enter a work request
    to have the MasterFlex tubing changed by the Instrumentation group.

# Troubleshooting

**[[PROBLEM 1:]{.smallcaps} Small number of Tips bent during run without
error]{.underline}**

If the tips are bent without error, the operator will have to snap the
method, raise the pod (if necessary), (see **b.** Moving Pod Via Manual
Control), replace bad tips with good ones and restart the snapped
method.

> 1\. Wait until there is no liquid in the tips.
>
> 2\. **Snap** the method by pressing the green snap button next to the
> stop button.
>
> ***NOTE:** The method will then be saved from this point (i.e. The
> Biomek will resume from the point that it was interrupted).*

a.  Determine if any of the plates being processed have been
    contaminated due to the bent tips (reagent on the top of the plate
    or plates being in wrong locations). If so, fail these plates in the
    database system. Scan them into the **Enter Fails or Comments** form
    in the **RCA DB**, select a failure mode, add an appropriate
    comment, and then click on submit.

    i.  Mark the plates that need to be failed with a sharpie so they
        will be removed from the production line later. Do not remove
        them from the deck or stackers at this time.

> ***NOTE:** Remember to select the **Do Not Submit** checkbox for any
> failed plates when the barcode validation window opens.*

ii. Print out a copy of these failed plates and give a copy to
    Sequencing Chemistry so that the plates can be resequenced. Write on
    the print out whether the plates are MB or ABI.

```{=html}
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```
b.  Moving the Pods via manual control during a run:

    i.  After the method is snapped, go to **TOOLS ** select **[manual
        control.]{.smallcaps}**

> ***NOTE:** You will not be able to manually move the pods in the
> middle of a run. You must **SNAP** the method first.*

ii. Select **Advanced controls.**

iii. Select **Pod 1** (if tip problem)/ **Pod 2** for gripper errors.

```{=html}
<!-- -->
```
c.  To move the pod up, select **Move Z-Max** (you may also click or
    select the up arrow button to move the pod upwards).

> ***NOTE**: Z-Axis moves the pods up and down, Y-Axis moved the pods
> back to front, and X-Axis moves the pods left to right.*

d.  Check to see if tips are bent.

e.  If necessary, replace any bent tips and/or reposition any plates.

    i.  Locking pliers can be used to remove bent tips.

    ii. Replace the bent tips with new ones from the **SPARE TIPS** box.

f.  Close the Advanced Control (Close) and Manual Control (Exit)
    windows.

g.  Select the **RUN** button (green play button) and the method will
    resume.

**[[PROBLEM 2: LARGE NUMBER OF TIPS BENT DURING RUN (WITH ERROR --
LIQUID IN TIPS)]{.underline}]{.smallcaps}**

If the Biomek crashes while liquid is loaded in the tips, the operator
must abort the method, unload the bent tips and start the run over from
the beginning of the current step.

1.  Abort the run by selecting the Abort button on the error window.

2.  Unload the bent tips: Go to **File  New Method  Run (or File 
    Open  Unload Tips)**. This will unload the bent tips into the tip
    box.

3.  Remove the old tip box from the deck and dispose of in autoclave
    bag. Replace with a new tip box (Label with your *initials and
    date*). Perform a tip QC now or some time that same day by following
    the **QC Protocol for the Biomek FX Liquid Handler**.

4.  *Instrument crash during BET addition:* The operator should put
    dummy plates into Stacker A to replace those that already have BET
    added to them. Run the normal method, but put the original plates on
    the deck after BET is added to the dummy plates.

5.  *Instrument crash during EtOH Wash:* The operator should put dummy
    plates into Stacker A to replace those that have already been
    washed. Run the program **ETOH_Wash_and \_Transfer_SW_091604.bmt**.
    After the dummy plates have been washed, put the original plates on
    the deck.

6.  *Instrument crash during Final Transfer:* The operator should put
    dummy plates into Stacker A and C to replace those that have already
    been transferred. Run the program **Resuspension and
    Transfer_SW_091604.bmt**

```{=html}
<!-- -->
```
a.  If neither the source plate nor the destination plates have been
    scanned, the operator must put the original plate on the deck after
    the dummy plate has been transferred, but before it is scanned.

b.  If the source plate has been scanned but not the destination, the
    operator must put both the source and destination plates on the deck
    after the dummy plate transfers.

c.  If the source and destination plates have been scanned, the operator
    can remove these plates from the set to be run with the **Final
    Resuspension and Transfer** program.

**[PROBLEM 3: [LARGE NUMBER OF TIPS BENT DURING THE RUN (WITH ERROR --
NO LIQUID IN TIPS)]{.smallcaps}]{.underline}**

If the Biomek crashes with no liquid in the tips, the operator can
change the tips and continue from where the instrument left off.

1.  **Snap** the current method by pressing the green snap button next
    to the stop button.

2.  Unload the bent tips: Go to **File  Open 
    unload_tips_midrun_SW_051004.bmt**.

3.  Run this method. This will unload the tips, but not clear the world
    file of all plate locations.

4.  Remove the old tip box from the deck and dispose of in autoclave
    bag. Replace with a new tip box (Label with your *initials and
    date*). Perform a tip QC now or some time that same day by following
    the **QC Protocol for the Biomek FX Liquid Handler**.

5.  Reload the new tips: Go to **File  Open 
    reload_tips_midrun_SW_051004.bmt**.

6.  Run this method. This will load and wash tips without clearing the
    world file of plate locations.

7.  Go to **File  Load continuation**. This is your method continuation
    that was created by the snap method.

8.  Press **RUN**.

**[PROBLEM 4: PLATE CARRIED TO WASH STATION]{.underline}**

1.  If the pod carries the plate to the wash station, **snap** the
    method.

2.  Use the manual controls *(Refer to Problem 1)* to move the pod up
    allowing you to move the pod up from the wash trough.

3.  Verify that the plate has not been contaminated (look for uneven
    well volumes and possibly splashing on the top of the plate.) If you
    think this plate was damaged or contaminated, mark it with a sharpie
    (so that you can fail it at the end of the run and place it in the
    appropriate position upon the deck.) If needed, replace any bent
    tips that could be catching on the plate. Then resume the method.

> ***NOTE:** Remember to select the **Do Not Submit** checkbox for any
> failed plates when the barcode validation window opens.*

**[PROBLEM 5: GRIPPER ERROR]{.underline}**

1.  Sometimes a gripper error occurs saying that the "gripper failed to
    retract" or that the "gripper failed to extend." When this happens,
    hit retry on error prompt window. You may need to do this several
    times.

2.  If this does not work, you must abort the run.

3.  Go to Manual Controls  select **Advanced Manual Control**  Select
    **POD 2** click on **Retract Gripper**  reload the plates and
    restart the run.

4.  Put a work request into the system to inform Instrumentation of the
    problem.

**[PROBLEM 6: LIGHT CURTAIN ERROR]{.underline}**

This type of error occurs when the light curtain is broken. When this
happens, the run will stop and there will be a RED error window prompt.
Click **OK**. This will resume the program from where it was
interrupted.

**[PROBLEM 7: UNLOCKING CLAMPS ON MAGNETS AND PLATE
FLATTENERS]{.underline}**

During the run, if a plate needs to be adjusted while on the magnet or
plate flattener, you can manually unlock clamps and fix the plate
problem.

1.  Press the pause button on the timer and either press the pause
    button on the Biomek software or break the light curtain to pause
    the run.

2.  To open the clamps, there is a switch located on the deck that says,
    *on/off*. Turning the switch to the *off* position will open the
    clamps.

3.  You can then remove or adjust plates.

4.  Turn switch to the *on* position.

5.  Resume the run by pressing the run icon or clicking **OK** on the
    light curtain error.

6.  Unpause the timer so that future errors will be sent to the pager.

**[PROBLEM 8: STACKER ERRORS]{.underline}**

1.  Press the pause button in the Biomek software (or break the light
    curtain) and pause the timer.

2.  Press the **Remote Local** button on the Stacker Carousel Pendant in
    order for the display to read "Keypad Control Only."

3.  Move the appropriate hotel to the shuttle by rotating the carousel
    either left or right, using the Stacker Carousel Pendant.

4.  Restack plates by moving the shuttle in (press **Shuttle In** on the
    keypad) and then moving the stack up (press **Stack Up** on the
    keypad).

5.  Ensure that all source plates are in order with their corresponding
    destination plates in Stacker C.

```{=html}
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```
a.  Plates that end up in Stacker B will be in the reverse order as
    those in Stacker C.

b.  Plates that end up in Stacker A will be in the same order as those
    in Stacker C.

    1.  Resume the run and unpause the timer.

    2.  If plates are being stacked crooked into the stacker notify
        instrumentation. The stacker may need to be leveled or the
        shuttle may be misaligned.

> ***NOTE**: Carefully examine plates that were stacked crooked to
> verify if the sample was splashed causing cross-contamination. If this
> occurs, fail the plate in venonat and request the Chemistry resequence
> the affected plates.*

**[PROBLEM 9: SUBMITTING PLATES TO THE DATABASE]{.underline}**

Sometimes a Biomek will have problems connecting to the database.

1.  First, try resubmitting the barcodes by clicking on **Connect to
    DB.** Submission may take awhile. There will be a prompt telling you
    that it is OK to submit the barcodes. Continue with submission as
    normal.

2.  If this does not work, you can manually submit the barcodes by
    following the troubleshooting steps below:

    a.  Press the ALT + F4 button to close the open barcode window.

    b.  Go to the desktop open up the folder **Shortcut to Logs**.

    c.  Look for the file with the name barcodes today's date and the
        time of the run in question. It should look something like this
        "**barcodes04-08-2004 15.45.21b.log**!"

    d.  Copy and paste the file or files in question onto a disk. You
        can do this by inserting a 3 ½ in floppy into the A drive open
        my computer and then drag from the shortcut folder into the A:
        drive.

    e.  Take the disk to another Biomek computer (select one that the
        internet connection is working.) Put disk into the A: drive open
        by going to my computer.

    f.  Drag the barcode file(s) in question into the **Shortcut to
        Logs** folder.

    g.  Right click on the barcode files in question select **Rename.**

    h.  Click after the "!", then remove the "!" from the file name and
        press **ENTER**. You will receive a prompt asking if you are
        sure you want to change the file name select **YES**.

> ***NOTE:** At this point the file name should look like:
> **barcodes04-08-2004 15.45.21b.log**.*

i.  In the shortcut to logs folder click on the "**up one level
    button**" (this button is a folder with an upward arrow). After
    selecting this you should see a folder called **GI Custom
    Programs**.

j.  Run the barcode-parsing program: Double click on the file named
    "**barcode_upload_biomek.exe."** (A shortcut to this program may be
    on the desktop of the computers operating the Biomeks)*.* A
    processed barcode file window will then pop up showing the
    association between the source plate and the ABI barcode verify that
    everything is correct and make necessary comments then select
    proceed and save as normal. This file will then be uploaded to the
    database and is now considered processed.

k.  Return to the original Biomek, open the **Shortcut to Logs** folder.

l.  Move the files that you just processed into the **Processed_logs**
    folder.

```{=html}
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```
3.  If the Biomek has scanned a portion of the barcodes in a run and the
    method is then snapped or restarted the Biomek software will
    generate multiple log files. This will cause the barcode-parsing
    software to present multiple windows to the user and often times the
    barcodes will be out of order. Follow the steps below to correct
    this problem:

    a.  Do not submit the barcodes to the database.

    b.  Press "**Alt+F4**" to close the barcode verification screen. If
        another barcode-parsing window opens, close it in the same
        manner.

    c.  On the desktop, open the **Shortcut to Logs** folder.

    d.  Find the file named "barcodes"; today's date and the time of the
        run in question it should look something like this
        "**barcodes04-08-2004 15.45.21b.log**!"

> ***NOTE:** This file should contain either the first or second portion
> of barcodes in the Biomek run.*

e.  Open the file; verify that the barcodes in this file are the ones
    that appeared on the barcode verification screen.

> ***NOTE 1:** In these logs the barcode parser program scans the first
> 4 source plates then the first 4 destination plates. So the first
> source plate scanned will then be paired with the first destination
> plate scanned.*
>
> ***NOTE 2:** You will most likely see that there are not enough
> entries to match the source and the destination to all of the plates.
> You will then need to copy these barcodes by highlighting them and
> pressing "**Ctrl+C**" (close this file by going to **File** and
> selecting **Close**).*

f.  You will then need to look for a second barcode log file in the same
    folder Short cuts to logs. It should have the same date as the
    previous file and approximately the same time. It will also end in
    an exclamation point. Open the file and verify that these are the
    remaining plates in the run. This second file might look like:
    **"barcodes04-08-2004 16.23.33.log!"**

g.  Paste the first set of barcodes into this file by pressing
    **"Ctrl+V"**. Keep the one space line at the top of the document.
    You should now have a file consisting of 4 scanned source plates,
    then 4 scanned destination (ABI) plates, then another 4 source
    plates, and another 4 destination plates and finally 2 source and 2
    destination. Verify that the scanning order is correct by checking
    the time that each scan took place.

h.  Save this by clicking **File**, then **Save**. Then exit out of the
    file screen. By going to **file** and **Close**.

i.  The file now needs to be renamed. In the **"shortcut to logs"**
    folder find the file in that has been modified. Right click on the
    title with the Mouse select **Rename.** The file name will then
    become highlighted and the cursor will be blinking. Remove the
    exclamation point from the file name.

j.  Move the un-modified file (barcodes04-08-2004 15.45.21b.log! in this
    case) to the **"Processed_Logs"** folder.

k.  Go to the desktop and open the **shortcut to barcode upload
    program.** The barcodes from the batch in question should appear in
    the correct order and appropriately matched. Verify then Proceed as
    normal by selecting **Proceed**, **entering the batch info** and
    **Save.**

**[PROBLEM 10: Home all axes]{.underline}**

Each axis (X, Y, Z & D) must be homed when the Biomek power is turned
off and on.

1.  Go to the **Tools** menu  select **Manual Control**  select **Home
    all Axes**.

2.  A window will open asking if there are any tips on POD1. If so, they
    will be shucked. Unload tips back into tip box by running the
    **unload_tips_midrun** method before continuing.

3.  Click **OK**. Stop button will become available.

4.  Click **Exit** when machine is stationary.

# 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

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

**5/27/04** -- Damon Tighe and Helene Feil performed an audit between
8:30AM -- 12:30PM. The operator was Catherine Gordon.

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

**7/26/04 --** Initializing Biomek Carousal and Homing All Axes.

**8/17/04** -- No longer need to spin down ABI destination plates after
Standard has been added. We now visually check every plate after
standard has been added and seal the plates. We only spin down if there
are bubbles or well volumes don't look consistent.

**12/12/04 --** Start taking tip QC's daily at 10pm (the start of the
3^rd^ shift), or immediately after the tips have been changed(PCN).

**1/14/05-**. We quit dispensing standard in the A1 well (12/20/04) of
the ABI plates. Mark unpaired plates with a blue "S" (12/22/04) and
place these plates in a bin marked "unpaired" for the appropriate
platform.

Updated program names that we are currently using on the biomek.

02/16/05- Begin taking weekly tip QC's instead of daily. QC will occur
at the end of 2^nd^ shift or the beginning of 3^rd^ shift at 10pm every
Wednesday. (PCN)

**5/10/05** -- Changes to Ismatec tubing shift and change. Too much
lubrication was being used and causing premature tears in tubing. SOP
changed so that less lubricant is used & pump rollers are no longer
sprayed.

**ADDENDUM**

**[Cybio Print & Apply Addendum to Post sequencing BET cleanup for ABI
plates:]{.underline}**

1.  Follow steps 1.0 thru 2.2a on the Bet Post sequencing BET Protocol.
    After scanning the source plates into the database, load them into
    stacker A of the biomek. Load the same number of pre-barcoded ABI
    plates into stacker C of the Biomek and start the current program.
    If unclear on how to start the biomek refer to Section 3.0 Biomek FX
    Setup of the Protocol.

2.  After the biomek has finished the run be sure to save and upload the
    barcodes in the Barcode Submission program displayed at the end of
    each run on the biomek. Discard the source plates from stacker B,
    and remove the ABI destination plates. Seal the plates with the
    Velocity 11 plate sealer (with the pierceable seal.)

***NOTE**: Do not remove the source or destination plates from the
Biomek FX before they have been uploaded to the database by the Barcode
Submission program.*

3.  After sealing these plates they are now ready to be labeled using
    the Cybio print and apply. Stack up to 40 ABI plates in the Cybio
    stacker labeled Load 1. If there are more than 40 plates, load up to
    40 additional plates in the stacker labeled Load 2. The blank side
    of the plate should be facing out of the stacker; this is where the
    barcode will be applied (The ABI barcode will be loaded facing the
    back of the stacker.)

> **Note:** You cannot keep loading or load more than 40 plates into
> each stacker. If more than 40 plates are placed into either load 1 or
> load 2 stackers the Unload stackers will overflow. Possibly allowing
> plates to fall.

1.  Open the Cybio program by doubling clicking on the Icon on the
    desktop labeled Cybio Control.

2.  Then go to **FILE→Local Disk C:→Cybio Software→2005.01.24 BG Two
    Stack short Labels.** (Below is the opening screen of the program)

![](media/image1.png){width="5.995138888888889in"
height="3.951388888888889in"}

3.  To start the Cybio You can either go to
    E[X]{.underline}ECUTE→Program [E]{.underline}xecute (F5) or click on
    the icon with an arrow going down with lines across. (It is the
    15^th^ icon from the right side)

4.  After selecting this icon the following message will be displayed as
    the cybio initializes. The shuttle will move first to the right and
    then to the left of the track.

![](media/image2.png){width="3.3430555555555554in"
height="1.5833333333333333in"}

Wait for a moment. After the Cybio has finished initializing the
following prompt will be displayed when the shuttle is in the far left
position.

![](media/image3.png){width="1.9270833333333333in" height="1.3125in"}

Verify that the shuttle is empty then select Ok.

5.  The Cybio will now begin removing the plates from the stacker
    labeled Load 1, scanning the plate, affixing the barcode, and
    restacking the plates into the Unload stackers. After the plates are
    stacked into the unload stacker with the label affixed to the side
    of the ABI plate, the operator can then unload them and mark the
    plates with the appropriate marking according to the Color code
    chart posted through out the lab and place these plates in the ABI
    backlog refrigerator in Room 141.

> ***Note:** If the Cybio fails to label any of the plates try replacing
> the plate in the load stacker for it to label the plate again. If this
> fails verify that all of the barcodes have been submitted to the
> Database on the biomeks. Sometimes there is a lag time after
> submitting the barcodes to the database and the Cybio accessing this
> information. If this occurs hold the plates in the TEG backlog and
> wait for 15 mins to an hour and try again. If it still fails enter a
> work request.*
>
> ***Note:** If the Cybio crashes while running and the operator has to
> restart the program, the first stacker Load 1 must be refilled with 40
> plates if you want the program to go to the second stacker (Load 2).*

6.  When the program is finished a message will appear stating that
    program has been executed without errors.

> ![](media/image4.png){width="2.7493055555555554in" height="1.3125in"}
>
> ***Note:** If only 40 plates are loaded into Load 1 you will receive
> the following message. This only means that it was unable to get a
> plate from Load 2.*
>
> ![](media/image5.png){width="2.7708333333333335in"
> height="1.3854166666666667in"}