Rolling Circle Amplification

**on Plasmid Glycerol Stock**

Automated Process (Matrix PlateMate Plus)

> Version Number: 3.4
>
> Production Start Date: 10/01/03
>
> Version 3.4 Date: 10/22/04
>
> Authors: Arshi Khan, Nancy Hammon, Christine Lansang
>
> Reviewed/Revised by: Danielle Mihalkanin, Lena Philip

# Summary

In preparation for sequencing, TempliPhi is used to amplify the plasmid
DNA template. This is a 4-2-4 reaction (4μl of 1X denaturation buffer,
2μl glycerol stock sample, and 4μl TempliPhi Premix).

# Materials & Reagents

+----------------------------+---------------------------+-------------+
| **[Materials/Reagent       | **[Vendor]{.underline}**  | **[Stock    |
| s/Equipment]{.underline}** |                           | Number]{.u  |
|                            | Axygen/ISC BioExpress     | nderline}** |
| *                          |                           |             |
| [Disposables]{.underline}* | Matrix                    | T-3059-1CS  |
|                            |                           |             |
| 384-well PCR Microplate,   | Velocity 11               | 5302        |
| Clear                      |                           |             |
|                            | Corning/VWR               | 06643-001   |
| 12.5-μl 384 Automation     |                           |             |
| Tips                       | Beckman                   | 430290      |
|                            |                           |             |
| PlateLoc Peelable Heat     | Edge BioSystems           | 538619      |
| Seal                       |                           |             |
|                            | Amersham Biosciences (GE  | 48461       |
| 50-ml Centrifuge Tube      | Healthcare)               |             |
|                            |                           | 25-6400-01  |
| Aluminum Foil Lids         | Monahan Paper Company     |             |
|                            |                           | 775011      |
| Clear Adhesive Plate       | AAPER Alcohol (LBNL)      |             |
| Sealers                    |                           | 6810-46175  |
|                            | Millipore Milli-Q System  |             |
| *[Reagents]{.underline}*   |                           | \-          |
|                            | Thermo Electron           |             |
| TempliPhi 10,000 Reaction  |                           | \-          |
| Kit:                       | Thermo Electron           |             |
|                            |                           | 24073290    |
| TempliPhi PreMix (5        | Titertek                  |             |
| bottles)                   |                           | 0978521022  |
|                            | Matrix                    |             |
| 1X Denaturation Buffer (5  |                           | \-          |
| bottles)                   | Perkin Elmer (Applied     |             |
|                            | Biosystems)               | \-          |
| pUC19 Control DNA (1 vial) |                           |             |
|                            | Velocity 11               | \-          |
| Pure Bright Germicidal     |                           |             |
| Bleach                     | Eppendorf                 |             |
|                            |                           |             |
| 95% Ethanol (200 Proof)    |                           |             |
|                            |                           |             |
| Milli-Q Water              |                           |             |
|                            |                           |             |
| *[Equipment]{.underline}*  |                           |             |
|                            |                           |             |
| 96/384-well Multidrop      |                           |             |
| Micro                      |                           |             |
|                            |                           |             |
| Multidrop Micro cassettes  |                           |             |
| (plastic tip)              |                           |             |
|                            |                           |             |
| Multidrop Micro cassettes  |                           |             |
| (metal tip)                |                           |             |
|                            |                           |             |
| PlateMate Plus             |                           |             |
|                            |                           |             |
| GeneAmp PCR System 9700    |                           |             |
|                            |                           |             |
| PlateLoc Thermal Plate     |                           |             |
| Sealer                     |                           |             |
|                            |                           |             |
| Centrifuge 5461            |                           |             |
+----------------------------+---------------------------+-------------+

# 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

**[Multidrop Micro]{.underline}**

NOTE 1: This should be done prior to dispensing 1X denaturation buffer
into the 1^st^ plate of each 36-plate batch that is processed and
TempliPhi into the 1^st^ and 36^th^ plates of each 36-plate batch that
is processed.

NOTE 2: Make sure to use Multidrop Micro 1 to dispense TempliPhi and
Multidrop Micro 4 to dispense buffer.

NOTE 3: Make sure the Multidrop Micro is set for a 384 plate, 4µl
volume, and 24 columns (col).

NOTE 4: Refer to the **Multidrop Micro** in the **Instrument Maintenance
& Settings** at the end of the protocol for proper Priming, Dispensing,
and Purging techniques.

1.  Prior to dispensing any liquid, go to the balance and tare the
    384-well Axygen plate (empty Axygen plate for buffer, and
    destination Axygen plate containing 4µl Buffer and 2µl Glycerol
    Stock for TempliPhi ).

2.  Dispense the appropriate amount of buffer or TempliPhi.

3.  Visually check to make sure that the reagent was dispensed into all
    384 wells and that the well volumes are uniform.

4.  Return to the balance and obtain the new weight.

5.  If weights are all within the range continue to dispense all the
    plates in the batch. If not, set the bad cassette aside for the
    Instrumentation group and change to a new cassette (after performing
    a calibration check on the new cassette).

    a.  Acceptable weight range after 4μl 1X denaturation buffer
        addition: **1.409g-1.697g**.

    b.  Acceptable weight range after 4μl TempliPhi addition:
        **1.409g-1.678g**.

6.  Record these values in the buffer or RCA (TempliPhi) calibration
    excel spreadsheet.

> ***NOTE 1:** To open excel spreadsheet click on **Shortcut to MD
> Calibrations** on desktop. If shortcut is unavailable go to **Network
> Places** on desktop and click on **Entire Network** click on **Entire
> Contents** click on **Microsoft Windows Network** click on **jgi_psf**
> click on **Octopus** click on **ProdSeq** click on **RCA** click on
> **Calibration** click on **Multidrop Calibrations** click on **daily
> MD calibrations** select **Buffer** or **TempliPhi** worksheet.*
>
> ***NOTE 2:** If the weight entered in the excel spreadsheet is too
> close to the high or low range, the number entered in the weight
> column will be highlighted in a red box and the text **WARNING** will
> appear in orange under the Spec. column. If this occurs, continue to
> use the cassette but monitor it carefully so that it does not fall out
> of range. If it is continuously high or low and in the WARNING zone,
> contact your supervisor to determine how to proceed.*
>
> ***NOTE 3:** If the weight entered in the excel spreadsheet falls out
> of the range, the number entered in the weight column will be
> highlighted in a red box and the text **DO NOT USE** will appear in a
> red box under the Spec. column. If this occurs, set the bad cassette
> aside for the Instrumentation group and change to a new cassette
> (after performing a calibration check on the new cassette).*

**[PlateMate Plus]{.underline}**

[Dye QC:]{.underline}

A tip QC should be performed using blue dye and the Spectramax 384 Plus
twice per day (morning and end of the day). Refer to the **QC Protocol
for the PlateMate Liquid Handler**. Tips should only be changed if the
tip QC fails, if there is any clogging, or if there is tip
contamination.

[Gravimetric QC]{.underline}:

> ***NOTE 1:** This should be performed on one plate within the first 10
> plates of first and last batch per day per Platemate Plus at **step
> 4.13**.*

1.  Without disturbing the Platemate, retrieve an empty axygen
    destination plate from Right Stacker B.

2.  Place the empty plate on the weighing balance and press the **TARE**
    button.

3.  Return the tared empty plate back to Right Stacker B.

4.  After the tared plate has been aliquoted, remove it from Right
    Stacker A and replace it with an empty Axygen plate.

5.  Reweigh the aliquoted plate on the weighing balance.

6.  Enter the weight into the Plate Weight Excel Spreadsheet.

> ***NOTE 1:** To open the Excel spreadsheet, click on **Shortcut to
> Platemate Daily Calibration** on desktop. If shortcut is unavailable
> go to Network Places on desktop and click on Entire Network click on
> Entire Contents click on Microsoft Windows Network click on jgi_psf
> click on Octopus click on ProdSeq click on RCA click on Calibrations
> click on Platemate Calibration click on Platemate Daily Calibration
> excel sheet.*
>
> ***NOTE 2**: There is a tolerance range. If it out of the range
> specified on the excel sheet, record the plate weight take another
> plate weight from a subsequent plate. If it fails the second time,
> notify a supervisor or QC within the RCA mail document.*

7.  Remove the empty Axygen plate from Right Stacker A on the Platemate
    and replace it with the aliquoted weighed plate.

8.  Repeat steps **1 -- 7** with a destination plate on the other
    platemate.

9.  Click **Save** on the Excel spreadsheet.

# Procedure

NOTE 1: All reagents/stock solutions should be prepared prior to the
start of the procedure.

NOTE 2: Make sure to wear gloves throughout the entire RCA procedure.

NOTE 3: Print out the daily Inoclist in order to determine which
glycerol stocks need to be pulled out for the RCA reaction and what
their primer association will be.

**1. [Sample and Reagent Preparation]{.underline}**

1.  Remove the glycerol stock plates from the 4^o^C deli in Rm140
    (Tuesday-Friday) or Freezer 014 in Rm139 (Monday). Pull one batch
    out at a time. Glycerol stocks can stay at room temp for no more
    than 4 hours. Keep in 4^o^C deli while not in use.

2.  Thaw the 384-well glycerol stocks at either: room temperature for 1
    hour, 4C overnight, or at 30 -- 37C for a maximum of 30 minutes.

3.  Remove the TempliPhi 10,000 reaction kit from -80C storage freezer.

> ***NOTE:** One TempliPhi bottle dispenses into approximately 12
> plates.*

4.  Remove the TempliPhi premix from the kit and place it in an
    ice-water bath until it has completely thawed. Make sure that the
    temperature remains at 0 -- 4^o^C.

> ***NOTE 1:** TempliPhi may take up to 4 hours to thaw.*
>
> ***NOTE 2:** An electric thermometer is used to monitor the batch
> temperature. The QC group should calibrate the thermometer every 6
> months.*

5.  Remove the denaturation buffer from the kit and place it on the
    counter until the end of the day.

6.  Store the pUC19 control DNA in the -80C storage freezer located in
    Rm 140.

> ***NOTE:** The pUC19 will not be needed for the RCA reaction*.

**2. [PlateMate Startup]{.underline}**

> ***NOTE:** Make sure fresh 2% bleach and 70% ethanol are prepared
> prior to proceeding.*

1.  Turn on the PlateMate robots and allow for homing procedure to
    finish.

2.  Fill the water supply reservoir with Milli-Q water and check to make
    sure that the waste reservoir is empty.

> ***Checkpoint 1:** Make sure that the tubing is in the waste
> reservoir.*
>
> ***Checkpoint 2:** Make sure the quick disconnect fitting is pushed in
> all the way on the Milli-Q water supply reservoir.*

3.  Add no more than 70mL fresh 2% bleach to an empty Stage 2 reservoir.
    *Refer to Figure 1 in Appendix A*.

4.  Open the **PlateMate Plus** program on the computer located next to
    the PlateMate.

5.  If this is the first PlateMate run of the day:

    a.  Load the pipette tips:

        i.  Go to the **Add-Ins** menu select **Change Pipettor** **or
            Tips** select **Replace Pipettor or Tips** select **Tips
            only**:

            1.  Check *Insert Tip Magazine*.

            2.  Check *Home Pipettor*.

        ii. Click on **START**.

        iii. Insert the tip magazine.

        iv. Click on **DONE**.

        v.  Click **OK**.

    b.  Prime the wash station:

        i.  On the manual control keypad located behind the PlateMate,
            press **NEXT**.

        ii. Press **2**: *Head*.

        iii. Press **3**: *Stage 3*.

        iv. Hold down **0**: *W_P* until water starts flowing through
            the wash station*.*

        v.  Press **RESET**.

    c.  Perform a tip QC (Refer to **PlateMate Plus** under the
        **Calibration Check** section).

**3. [Dispensing of Denaturation Buffer]{.underline}**

1.  Install the Multidrop Micro cassette and prime with Milli-Q water
    (Refer to the **Installation** and **Priming** instructions under
    the **Multidrop Micro Instrument Maintenance** section).

> ***NOTE 1:** Make sure to use fresh clean autoclaved water bottles
> daily.*
>
> ***NOTE 2:** Make sure to use separate bottles for the RCA & Buffer
> water reservoirs.*

2.  Remove the 1X denaturation buffer from the 4^o^C deli located in Rm
    140.

3.  Pour the 1X denaturation buffer into a 50-ml centrifuge tube
    labeled: *buffer, lot#, date*.

4.  Using **Multidrop Micro 4**, dispense **4μl** of 1X denaturation
    buffer into each well of a new 384-well PCR plate for one batch of
    36 plates (Refer to the **Priming** and **Dispensing** instructions
    under the **Multidrop Micro Instrument Maintenance** section).

> ***NOTE 1:** Perform a calibration check on the first plate of each
> batch by referring to the **Calibration Check** section on page 2.
> Acceptable range 1.409g-1.697g.*
>
> ***NOTE 2:** Buffer plates should not be prepared more than 30 minutes
> ahead of time.*
>
> ***NOTE 3:** Monitor the 50-ml tube to ensure that there is enough
> buffer for the plates.*
>
> ***NOTE 4:*** *If a clog develops or some other Multidrop Micro
> cassette failure occurs in the middle of dispensing, stop processing
> the batch with the bad cassette and continue processing the batch with
> a new cassette (after performing a calibration check on the new
> cassette). Make sure to note the change in the calibration excel
> spreadsheet.*

5.  Visually check to make sure that 1X denaturation buffer was
    dispensed into all 384 wells and that the well volumes are uniform.

> ***Checkpoint:** If 4 wells or less per plate are empty, manually add
> 4μl of 1X denaturation buffer to the empty wells with a pipette. If
> more than 4 wells are empty or overfilled, re-aliquot the plate with
> the Multidrop Micro. If there is something wrong with a tip (e.g.
> clogged tip), submit a work request and change the Multidrop Micro
> cassette.*

6.  Once you are done dispensing the buffer, empty the buffer back into
    the 50-ml tube, clean the buffer cassette with Milli-Q water, and
    remove the cassette from the Multidrop Micro (Refer to the
    **Purging**, **Cleaning cassette and trough**, and **Removing
    cassette for storage** instructions under the **Multidrop Micro
    Instrument Maintenance** section).

> ***NOTE 1:** The buffer cassette will be used daily until it falls out
> of calibration or a clog develops.*
>
> ***NOTE 2:*** *If the buffer cassette will be used for another batch
> the cassette does not need to be removed, instead release the tubing
> only.*
>
> ***NOTE 3:** Always empty the waste from the trough into a waste
> beaker.*

7.  Place the buffer back in the 4^o^C deli located in Rm 140.

8.  Place the plates in the centrifuge for a quick spin **(1 minute at
    1,000 rpm)**.

> ***NOTE:*** *If the buffer plates are not going to be used within 5-10
> minutes, seal the top plate of the stack with a clear seal, label with
> the batch \# and PM #, and store the plates in the 4^o^C deli located
> in RM 140. Plates should sit in the deli no longer than 30 minutes.*

**4. [Aliquoting 2μl of Glycerol Stock ]{.underline}**

1.  Carefully unseal the thawed glycerol stock plates. Keep the primer
    types together.

> ***NOTE 1:*** *Visually check the glycerol stock plates to see if
> there are any holes or irregularities such as contamination. If there
> are any holes or irregularities, return the glycerol stock plates to
> the Libraries supervisor so that the plate may be fixed or replaced.*
>
> ***NOTE 2:*** *If the glycerol stock plates are not going to be used
> within 5-10 minutes, seal the top plate of the stack with a clear
> seal, label the top seal with PM \# and batch #, and store the plates
> in the 4^o^C deli located in Rm 140.*

2.  In the Venonat database
    ([**http://venonat.jgi-psf.org/psf/PSF.home**](http://venonat.jgi-psf.org/psf/PSF.home)),
    click on **RCA DB** **STEP 1 - Scan in Library Plates to be
    Transferred**:

```{=html}
<!-- -->
```
a.  Select the correct information from the drop-down menus: *Operator,
    Buffer Lot, Transfer Instrument, Denature Instrument, RCA
    Instrument, RCA Lot*, & *Daily Batch Number.* Using the barcode
    scanner, scan in the *Buffer Cassette and RCA Cassette*.

> ***NOTE:** For Transfer Instrument, select Platemate 1A instead of
> Platemate 1. Platemate 1A is the designation in the database for the
> new dispense program.*

b.  Use the barcode scanner to scan in a batch of glycerol stock plates
    (18 plates per PlateMate).

c.  Click on **SUBMIT**.

d.  Print the batch information page and place it in the RCA Run log
    binder.

> ***NOTE:** All information must be entered correctly into the
> database:*
>
> *i. If operator, buffer lot, transfer instrument, denature instrument,
> RCA instrument, or RCA lot are incorrect go to "Change Batch
> Information" in QC section of RCA DB, enter in the RCA Batch id# ,
> enter in all correct information and hit submit.*
>
> *ii. If buffer cassette, RCA cassette, or daily batch \# are incorrect
> go to "Change RCA Cassette, Buffer Cassette or Daily Batch Number by
> Individual Plate" in the QC section of RCA DB. Enter in all the
> correct information, scan in all plates affected and hit submit. If
> daily batch \# needs to be changed, in addition to DB change, change
> by hand on plate label itself. (re-mark label with sharpie)*

e.  If an error message appears, refer to the **Troubleshooting**
    section on how to requeue the plates.

    2.  Print the barcodes for the destination plates.

    3.  Mark the barcodes with the correct primer association color.

> ***NOTE:** The primer association color key is located on the RCA
> countertop.*

4.  Affix the destination barcodes onto the 384-well plates containing
    buffer (on the indented side of the plates).

5.  In the Venonat database, click on **RCA DB** click on **New RCA
    Plate Check** enter in the batch ID from the page printed in step
    **4.2.d**. click on **SUBMIT** scan in the destination plates click
    on **SUBMIT**.

> ***NOTE 1:** If there is an error, an incorrect message error will
> appear. You must then manually rearrange the destination plates to put
> them in the correct order as the source glycerol stocks.*
>
> ***NOTE 2:** Prior to loading the stackers, visually check to see that
> the stack of glycerol plates corresponds to the stack of destination
> plates.*

6.  Open the **PlateMatePlus** software program on the computer desktop
    select **Files** select **Open**

    a.  For PM 1: Select
        **Glycerol_Stock_Transfer_into_4ul_Buff_SW_112204.cms**.

    b.  For PM 3: Select
        **Glycerol_Stock_Transfer_into_4ul_Buff_SW_072704.cms**.

7.  Change the number of loop iterations on both transfer groups to
    correspond to the number of glycerol stock plates for transfer and
    click on the **SET** button.

8.  Load the glycerol stock plates (source plates) into Left Stacker B
    with the A1 well in the top left corner. *Refer to Figure 1 in
    Appendix A*.

9.  Load the 384-well plates (destination plates) containing denature
    buffer into Right Stacker B with the A1 well in the top left corner.
    Be sure to match the source and destination plates in the same
    order. *Refer to Figure 1 in Appendix A.*

10. Click on the **PLAY** button and the program will automatically
    validate the sequence.

11. Click on the **START** button when ready.

12. Perform a gravimetric calibrationcheck of the Platemate according to
    the procedure in the **Calibration Check** section of the protocol.

13. Make sure that you continuously monitor the PlateMate to ensure that
    the water reservoir does not run low on water, the waste reservoir
    does not overfill, and no errors occur in production.

> ***NOTE:*** *A common error that occurs is either the glycerol plate
> or destination plate will not position correctly from the stacker to
> the stage. If this error occurs make sure both plates are aligned
> correctly on the stage and hit **Ignore** on the computer.*

14. After 18 plates, a pop-up message appears. Dump the bleach into a
    waste beaker, rinse the trough with Milli-Q water, add 70mL fresh 2%
    bleach to the trough, and click **OK**.

> ***NOTE:** When the bleach waste accumulates, it can be dumped down
> the sink.*

15. When the run is finished, remove the 384-well sample plates from
    Right Stacker A and the glycerol stock plates from Left Stacker A.

16. When the run is finished, the **Barcode Verification and
    Submission** program will open on the desktop.

    a.  If the window is gray, then all barcodes were successfully
        scanned and no mix-ups occurred. Fill in the requested
        information and submit.

    b.  If the window is yellow, the barcode scanner did not scan one or
        more plate barcodes. Carefully identify the plates that need to
        be scanned into the form (matching source and destination
        plates) and scan them manually using the handheld scanner. Fill
        in the requested information and submit.

    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 by
        checking the **Do not submit** box next to the affected plates,
        filling in the requested information and submitting. These
        plates must also be scanned into the **Enter Fails or Comments**
        form in the **RCA DB** as failures, & an Incident Report should
        be filed

17. Place the 384-well destination plates in the centrifuge for a quick
    spin (**1 minute at 1,000rpm**).

> ***NOTE:** If time does not permit you to do steps **5** and **6**,
> seal the top plate of the stack with a clear seal and store the plates
> in the 4^o^C deli located in RM 140. The plates can sit in the deli
> for up to **one hour**.*

18. Seal the 384-well sample plates with the PlateLoc sealer.

19. Seal the glycerol stock plates with aluminum foil lids using the
    automated pneumatic sealer.

> ***NOTE:** The glycerol stock plates do not need to be sealed
> immediately following step 4.16, given time constraints. If the plates
> cannot be sealed right away, place an aluminum foil seal on the top
> plate of the stack, label with the batch #, and set the plates on the
> countertop until there is sufficient time to seal the plates
> properly.*

20. Rerack the glycerol stock plates and return them to the -80C freezer

**5. [Plates for Stamping QC]{.underline}**

1.  Using the Innoc List for the current RCA date, choose 1 glycerol
    stock plate from 4 different libraries from each vector. (If there
    is only one library, randomly select 4 plates from that library to
    stamp.)

2.  Make sure the libraries selected will represent the production line
    appropriately; first - large genomes and clones, then microbes or
    BACs.

3.  Place the glycerol stock plate with lid in a bin labeled, "For
    Stamping QC" in the 4˚C deli.

> ***NOTE**: These plates should have already been processed on the
> Platemate in the RCA process.*

1.  **[Heat Lysis]{.underline}**

    1.  In the Venonat database, click on **RCA DB** [**STEP 2 -Link
        Plates with Cyclers for
        Denaturation**](http://venonat.jgi-psf.org/psfs/psf.wwv_media.show?p_type=plsql&p_id=6805&p_currcornerid=2054&p_language=us)
        scan in the plates according to which side of the thermocyclers
        they will be loaded and click on **SUBMIT**.

    2.  Place the 384-well plates on their corresponding thermocyclers.

    3.  Run the **rca denature** program: *95C for 5 minutes, 4C
        forever*.

> ***NOTE 1:** Make sure that the thermocyclers are set for the user
> RCA.*
>
> ***NOTE 2:** On the thermocycler, once you have loaded the plates,
> close the lid, press any button if the screen is not on scroll to the
> **rca denature** program press **F1** to start the program.*

4.  After the program ends, remove the 384-well plates and immediately
    place them on ice.

> ***NOTE:** On the thermocycler, once the **rca denature** program has
> ended press **STOP** twice press **F5**.*

7.  **[Amplification with TempliPhi]{.underline}**

    1.  Install and prime the TempliPhi cassette.

> ***NOTE 1:** A new cassette will be installed daily for plastic tipped
> cassettes, when the cassette clogs or falls out of calibration for the
> steel tipped cassettes.*
>
> ***NOTE 2:** Perform a calibration check on the first and last plate
> of each batch. Acceptable weight range: **1.409-1.678g**.*

2.  Place a labeled (*TempliPhi,lot #, & date*) 50-ml centrifuge tube in
    a beaker of ice.

3.  Pour the thawed TempliPhi premix into the 50-ml centrifuge tube. If
    TempliPhi is approximately 95% thawed then gently swirl the
    TempliPhi to thaw the remaining chunks of ice otherwise keep thawing
    at 0-4C.

4.  Carefully peel the seals from each 384-well PCR plate.

> ***NOTE 1:** Remove the seals one at a time prior to dispensing
> TempliPhi into the plate.*
>
> ***NOTE 2**: If pieces of seals are stuck on plate, use a pipette tip
> or clear seal to remove them.*

5.  Using **Multidrop Micro 1**, dispense **4μl** of the TempliPhi
    premix into each well of the 384-well sample plate.

> ***NOTE 1:** Monitor the 50-ml tube to ensure that there is enough
> TempliPhi for the plates.*
>
> ***NOTE 2:*** *If a clog develops or some other Multidrop Micro
> cassette failure occurs in the middle of dispensing, continue
> processing the batch with a new cassette (after cleaning and
> performing a calibration check on the new cassette). Change the batch
> information in the database on all plates that will be using the new
> cassette: go to "Change RCA Cassette, Buffer Cassette or Daily Batch
> Number by Individual Plate" in the QC section of RCA DB. Enter in all
> the correct information, scan in all plates affected and hit submit.*

6.  Visually check to make sure that TempliPhi was added to all 384
    wells and that the well volumes are uniform.

> ***NOTE:** If TempliPhi was not added to all wells, then fail the
> plate in the **RCA DB** and inform your supervisor in order to
> determine if the plate needs to be resequenced*

7.  After dispensing TempliPhi into each plate, stack the plates in
    sterile bin labeled with the *batch #, date, and operator initials*,
    until TempliPhi has been dispensed into all of the plates.

> ***NOTE:** Spray bin with 70% EtOH and use Kimwipes to sterilize bin
> prior to use.*

8.  Place the plates in the centrifuge for a quick spin **(1 minute at
    1,000rpm)**.

9.  Use the PlateLoc Sealer to seal the 384-well sample plates.

10. Place the 384-well sample plates into the sterile bin stacked 4
    plates high in 9 stacks spread across the bin equidistant away from
    each other.

11. Label the bin with the *Time In* and the *Time the plates come out*
    then place the bin in the 30C oven to incubate for 18 hours.

12. Purge TempliPhi from tubing, clean tubing with Milli-Q water, &
    remove the TempliPhi cassette from the Multidrop Micro (Refer to the
    **Purging**, **Cleaning cassette and trough**, and **Removing
    cassette for storage** instructions under the **Multidrop Micro
    Instrument Maintenance** section).

> ***NOTE 1:*** *If the TempliPhi cassette will be used for another
> batch, the TempliPhi cassette does not need to be removed. Instead,
> release the tubing only.*
>
> ***NOTE 2:** Always empty the waste from the trough into a waste
> beaker.*

13. Used cassette storage:

    a.  If using the plastic tipped cassettes, at the end of the last
        batch, return the TempliPhi cassette to the QC group.

    b.  If using the steel tipped cassettes, continue using the cassette
        in production until it clogs or fails calibration.

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8.  **[End of the Day Cleanup]{.underline}**

    1.  Pour the remaining TempliPhi premix back into its original
        bottle and label: *date*. Place the TempliPhi premix on the
        bottom shelf of the --80^o^C freezer from where you obtained the
        TempliPhi 10,000 reaction kit.

    2.  Place the thawed denaturation buffer from step **1.5** into the
        4^o^C deli located in RM140.

    3.  Shutting down the PlateMate:

        a.  Perform a tip QC (Refer to **PlateMate Plus** under the
            **Calibration Check** section).

        b.  Remove the pipette tips:

            i.  Go to the **Add-Ins** menu select **Change Pipettor**
                **or Tips** select **Replace Pipettor or Tips** select
                **Tips only** enter in:

                1.  Check *Remove Tip Magazine*.

                2.  Check *Home Pipettor*.

            ii. Click on **START**.

            iii. Remove the tip magazine and store in the Matrix tip
                 box.

            iv. Click on **DONE**.

            v.  Click **OK**.

            vi. Turn off the PlateMate.

    4.  Empty the Milli-Q water bottle down the sink and store the empty
        bottle upside down.

    5.  Pour the waste down the sink.

    6.  Empty the bleach trough and rinse with Milli-Q water.

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9.  **[Heat Inactivation]{.underline}**

    1.  After the 18-hour incubation period, remove the 384-well sample
        plates from the 30C oven.

    2.  Determine which thermocyclers are operational.

    3.  In the Venonat database, click on **RCA DB** **STEP 3 -Scan in
        Plates for Final Heat Step** select the proper thermocyclers and
        scan in the plates and click on **SUBMIT**.

    4.  Place the 384-well sample plates on the thermocyclers in order
        of how they scan into the database.

    5.  Run the **rca95 final heat** program: *95C for 10 minutes, 4C
        forever*.

> ***NOTE 1:** Make sure that the thermocyclers are set for the user RCA
> or Chem.*
>
> ***NOTE 2:** On the thermocycler, once you have loaded the plates,
> close the lid, press any button if the screen is not on press **F1**
> scroll to the **rca95 final heat** program press **F1** press **F1**
> again.*

6.  Print out the FW and RV barcodes for each plate, mark the barcodes
    with the correct primer association color & place the barcodes in
    the Sequencing Chemistry bay.

7.  After the program ends, remove the 384-well sample plates and
    continue with the Big Dye or ET-Terminator sequencing chemistry
    protocols if this is the first batch of the day. If this is not the
    first batch of the day, store the plates in the 4^o^C deli located
    in RM 140.

> ***NOTE:** On the thermocycler, once the **rca95 final heat** program
> has ended press **STOP** twice press **F5**.*

# Reagent/Stock Preparation

**[2% Bleach]{.underline}**

***NOTE:** Prepare 2% Bleach daily in a fresh autoclaved bottle*

1.  Pour **490ml** of Milli-Q water into a 500-ml bottle.

2.  Add **10ml** of bleach.

3.  Mix well by inverting bottle.

4.  Label bottle: *2% bleach,* *date prepared and your initials*.

# Instrument Maintenance

**[Multidrop Micro]{.underline}**

**Switching on:**

Switch the instrument on by using the main power switch on the left-hand
side of the instrument. The green Power LED should light up.

**Installing the cassette:**

1.  Perform a visual check on the cassette and the cassette tubing to
    make sure that they are undamaged.

2.  Remove the tip protection from the dispensing cassette.

3.  Take the lower part of the cassette (with tips) into your right hand
    (the dispensing cassette tips should be pointing down) and the upper
    part in your left hand.

4.  Carefully place the eight tubes below the pump rotor and insert the
    lower part of the dispensing cassette into the lower part slots of
    the pump body. Check that the tubes are freely placed below the pump
    rotor and the tension limiting wires below the rotor shaft.

5.  Take a firm grip on the upper part of the dispensing cassette with
    your right hand and carefully pull the tubes around the pump rotor
    until the upper part reaches and fits into the upper part slots of
    the pump body.

6.  Check to make sure that both the upper and lower parts of the
    dispensing cassette are properly placed into their corresponding
    slots.

7.  Ensure that the tension limiting wires have a loose fit around the
    rotor shaft and tubes are evenly placed on the rotor needles (four
    tubes on each half of the pump rotor).

8.  Pull the rotor cover over the rotor.

9.  Remove the tubing weight protection.

**Priming:**

1.  After the cassette and priming trough are securely in place, place
    the tubing into a Milli-Q water reservoir.

> ***NOTE 1:** Fill fresh, clean, autoclaved bottles daily with MilliQ
> water.*
>
> ***NOTE 2**: Use separate wash bottles for buffer and RCA cassettes*.

2.  Press the **PRIME/DROP** key until all eight channels are dispensing
    continuously into the priming vessel.

3.  Visually check to make sure that all of the tips are dispensing
    evenly.

4.  Press the **EMPTY** key until no water is left in the cassette
    tubing.

> ***NOTE:** Make sure to periodically empty the trough into a waste
> container.*

5.  Place the tubing in the liquid that is to be dispensed and press the
    **PRIME/DROP** key until the liquid is dispensing evenly from all of
    the tips.

**Dispensing:**

1.  Check the control panel for *proper selection of plate type (96 or
    384 well plate), dispense volume, number of rows*.

2.  Place the 384-well plate onto the plate adapter. Make sure that it
    is seated properly.

3.  Press the **START** key to begin dispensing.

4.  Observe that all tips are dispensing properly (i.e. no clogs,
    missing the wells completely).

> ***NOTE:** If there is a clog in the tip, you must remove the
> cassette, submit a work request for the cassette, and install a new
> cassette.*

5.  Remove the plate and perform a visual check to make sure that the
    Multidrop Micro has dispensed accurately and evenly across wells.

6.  Repeat steps **2 -- 5** for each plate in the batch.

**Purging:**

1.  After all of the plates have been dispensed, press the **EMPTY** key
    to recycle any remaining liquid in the tubing.

2.  Remove the tubing from the reservoir.

**Cleaning the cassette and trough:**

1.  After you have finished dispensing, run Milli-Q water (trough full)
    through the tubing.

2.  At the end of the day, run Milli-Q water (3 troughs full) through
    the tubing.

3.  Empty and wipe the trough with a Kimwipe to ensure that no liquid
    gets on the sensor.

**Removing cassette for storage:**

1.  Push the cover off of the rotor.

2.  Remove the cassette from the Multidrop Micro.

3.  Dry any liquid remaining on the cassette with a Kimwipe.

4.  Replace the tip protection cover on the cassette.

5.  Replace the tubing weight protection.

6.  Place the cassette back in its corresponding box.

**Calibration Check:**

Refer to the **Calibration Check** section on page 2.

# Appendix A

Figure 1. PlateMate Setup.

+-------------+-------------+-------------+-------------+-------------+
|             |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
|             |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
| Glycerol    |             |             | Destination |             |
| Stock 4     |             |             | Plate 4     |             |
+-------------+-------------+-------------+-------------+-------------+
| Glycerol    |             |             | Destination |             |
| Stock 3     |             |             | Plate 3     |             |
+-------------+-------------+-------------+-------------+-------------+
| Glycerol    |             |             | Destination |             |
| Stock 2     |             |             | Plate 2     |             |
+-------------+-------------+-------------+-------------+-------------+
| Glycerol    |             |             | Destination |             |
| Stock 1     |             |             | Plate 1     |             |
+-------------+-------------+-------------+-------------+-------------+
| LEFT        | LEFT        | Pipettor    | RIGHT       | RIGHT       |
| STACKER B:  | STACKER A:  |             | STACKER B:  | STACKER A:  |
| START       | END SOURCE  | Head        | START       | END         |
| SOURCE      |             |             | DESTINATION | DESTINATION |
+-------------+-------------+-------------+-------------+-------------+

+----------------+-----------------+-----------------+-----------------+
| **STAGE 1**    | **STAGE 2**     | **STAGE 3**     | **STAGE 4**     |
|                |                 |                 |                 |
| **(Left        | **(2% Bleach)** | **(Wash         | **(Right        |
| Stackers)**    |                 | Station)**      | Stackers)**     |
+----------------+-----------------+-----------------+-----------------+

The PlateMate begins by bleaching and washing the tips. It then takes
the glycerol stock plate from Left Stacker B, and places it on stage 1.
The platform shifts over and stage 4 takes a 384-well plate with 1X
denaturation buffer out of the Right Stacker B. 2μl are then aspirated
from the glycerol stock plate and mixed into the 384-well plate with
denaturation buffer. The glycerol stock plate is then returned to Left
Stacker A and the 384-well plate is returned to Right Stacker A. Tips
are washed in a 2% bleach solution followed by a Milli-Q water rinse.

# Appendix B

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

**4/22/04 -- 5/07/04**: The *Multidrop Micro Cassette Handling Addendum*
to the protocol was implemented in production.

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

**5/26/04** -- Steve Wilson and Hope Tice performed an audit between
6:00AM --11:30 AM. The operator was Sako Boen.

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

**6/28/04 --** There is now a drop down menu to enter the RCA batch
number on the barcodes.

**6/29/04 -** The new cleaning procedure for the Multidrop Micro's is to
wash the cassettes with Milli-Q water following each batch and then with
Milli-Q water, 70% ethanol, and then a separate bottle of Milli-Q water
at the end of the shift.

**11/04** - After the templi-phi is added to the plates, they no longer
are put on ice. The plates are now stacked in sets of four when placed
in the incubator.

**1/5/05 --** Calibration weight ranges for Buffer & Templiphi have been
changed.

**2/18/05**- Added gravimetic calibration check for platmates and
changed dispense program on Platemate 1.

**05/25/05-** Began using steel tipped cassettes in production.

**06/6/05-** Eliminated ethanol wash of cassettes.

# Troubleshooting

**Step 4.2.e: After scanning the glycerol stock plates into the database
and clicking on SUBMIT, an error message appears, stating:** *ERROR!
\...\[error explanation\]...Please request a requeue if it needs to be
run again. Please notify your supervisor.*

> In the Venonat database, click on **RCA DB** **Scan Plates to
> Requeue** scan in the glycerol stock plates that were the source of
> the error click on **SUBMIT** Continue with step **4.3**.

If the problem persists, notify your supervisor.

**[Procedure for Thermal Cycler Recovery After a Power
Outage]{.underline}**

[Summary]{.underline}

This procedure details how to ensure thermal cycler recovery after a
power outage and how to handle production plates running on the cyclers
at the time.

> ***NOTE**: Action is based on Building Power Outage Duration*

[\<15secs]{.underline}

No action other than an inspection needs to be taken for an outage less
than 15 seconds long. The operator should check all the cyclers affected
to ensure the runs are continuing as required.

[\>15secs]{.underline}

If the power is off for 15 seconds or longer during a run, then the
method currently running will still restart. If the power fails while
executing a hold, or approaching a hold, then that hold temperature will
restart from the beginning.

[\>5 minutes]{.underline}

Turn off each cycler at the front power switch. Remove plates from the
cyclers and store them in the deli if the power is not restored in 5
minutes.

[\<2 hours]{.underline}

If building power is restored in 2 hours or less then put the plates
back onto the same cyclers and resume the run by turning each cycler
back on again at the front power switch. The operator should check all
the cyclers affected to ensure the runs are continuing as required.

[\>2 hours]{.underline}

If building power is not restored within 2 hours the plates should be
held for further testing.

> ***NOTE**: Any instrument that was idle prior to the power failure or
> did not automatically recover after an outage should be powered off
> and then back on after a 15 second pause.*

[PCR Network (Rm141) Recovery when Effected Runs are
Completed]{.underline}

-   Power off and then back on after a 15 second pause all the
    instruments in the network one at a time, wait for them all to reach
    the initial menu window.

-   Re-boot the PCR net computer and restart the PCR Network
    application.

-   Confirm that the PCR net software recognizes the units as they are
    returned to the system.

[Work Request]{.underline}

If required submit an Instrumentation Work Request for any instruments
continuing to have issues after the restoration of building power and
shutting off & on again.

# SOP Approval

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

  **Lab Supervisor**                              

  **Research &                                    
  Development**                                   

  **Instrumentation**                             

  **QC**                                          

  **Purchasing**                                  

  **EH & S**                                      

  **Informatics**                                 

  **Seq Assessment &                              
  Analysis**                                      

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