Cycle Sequencing using BigDye v3.1

**Performed on Rolling Circle Amplified DNA Template**

1/16^th^ Reaction (0.5BD)

> Version Number: 2.1
>
> Production Start Date: 10/01/03
>
> Version 2.1 Date: 05/06/04
>
> Authors: Arshi Khan, Nancy Hammon, Catherine Gordon
>
> Reviewed/Revised by: Amber Nivens

# Summary

The BigDye v3.1 sequencing kit is used to cycle sequence plasmid DNA,
which has been amplified by rolling circle amplification, in preparation
for sequencing on the ABI 3730xl instruments.

# Materials & Reagents

+---------------------------------+-----------------------+------------+
| **[Materials/Re                 | **[                   | **[Stock   |
| agents/Equipment]{.underline}** | Vendor]{.underline}** | N          |
|                                 |                       | umber]{.un |
| []{.mark}                       | Axygen/ISC BioExpress | derline}** |
|                                 |                       |            |
| *[Disposables]{.underline}*     | Corning/VWR           | T-3059-1CS |
|                                 |                       |            |
| 384-well PCR Microplate, Clear  | Velocity 11           | \-         |
|                                 |                       |            |
| 50-ml Centrifuge Tube           | Rainin                | 430290     |
|                                 |                       |            |
| PlateLoc Peelable Heat Seal     | Corning               | 06643-001  |
|                                 |                       |            |
| LTS Pipette Tips (2, 10, 100,   | Applied Biosystems    | 352076     |
| 200, 1000μl)                    |                       |            |
|                                 | IDT                   | 4336923    |
| 175 Graduated Conical tube with |                       |            |
| cap                             | IDT                   | \-         |
|                                 |                       |            |
| *[Reagents]{.underline}*        | Beckman Coulter       | PN 8546930 |
|                                 |                       |            |
| BigDye Terminator v3.1 Cycle    | AAPER Alcohol (LBNL)  | 6810-13289 |
| Sequencing Kit                  |                       |            |
|                                 | Millipore Milli-Q     | \-         |
| (with 5X BigDye Terminator Seq  | System                |            |
| Buffer)                         |                       | \-         |
|                                 | Robbins Scientific    |            |
| FW Sequencing Primer            |                       | \-         |
|                                 | Robbins Scientific    |            |
| RV Sequencing Primer            |                       | \-         |
|                                 | JGI                   |            |
| Coulter Clenz                   |                       | \-         |
|                                 | Rainin                |            |
| 100% Ethanol (200 Proof)        |                       | \-         |
|                                 | Perkin Elmer (Applied |            |
| Milli-Q Water                   | Biosystems)           | \-         |
|                                 |                       |            |
| *[Equipment]{.underline}*       | Velocity 11           | \-         |
|                                 |                       |            |
| 384-well Hydra Twister          | Pyrex                 | \-         |
|                                 |                       |            |
| Hydra Wash Reservoir            | Pyrex                 | \-         |
|                                 |                       |            |
| Cavro Dispensing Robot          | Miller-Stephenson     | \-         |
|                                 |                       |            |
| Pipettes (2, 10, 100, 200,      | Falcon/VWR            |            |
| 1000μl)                         |                       |            |
|                                 |                       |            |
| GeneAmp PCR System 9700         |                       |            |
|                                 |                       |            |
| PlateLoc Thermal Plate Sealer   |                       |            |
|                                 |                       |            |
| 250 ml, 500 ml bottles          |                       |            |
|                                 |                       |            |
| 600 ml beakers                  |                       |            |
|                                 |                       |            |
| PTFE Release Agent Dry          |                       |            |
| Lubricant                       |                       |            |
|                                 |                       |            |
| 125 ml double pharmaceutical    |                       |            |
| graduate                        |                       |            |
+---------------------------------+-----------------------+------------+

# 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

**[Hydra Twisters]{.underline}**

NOTE: This should be performed on one FW and one RV plate within the
first 10 plates per batch per Hydra Twister at step **2.12**.

1.  Without disturbing the Hydra Twister, retrieve an empty FW
    destination plate from Stacker 4.

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

3.  Return the tared empty FW destination plate back to Stacker 4.

4.  After the tared FW destination plate has been aliquoted, remove it
    from Stacker 5 and replace it with an empty Axygen plate.

5.  Reweigh the aliquoted FW destination plate on the weighing balance.

> ***NOTE 1**: If the weight ranges fall outside of **0.3366g --
> 0.3743g**, WARNING.*
>
> ***NOTE 2**: If the weight ranges fall outside of **0.3272g --
> 0.3837g**, DO NOT USE.*

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

> ***NOTE 1:** To open the Excel spreadsheet, click on **Shortcut to
> Plate Weight** on desktop.*
>
> ***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 Hydra 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, discontinue use of the
> hydra and submit a work request for calibration.*

7.  Remove the empty Axygen plate from Stacker 5 on the Hydra Twister
    and replace it with the aliquoted weighed FW destination plate.

8.  Repeat steps **1 -- 7** with a RV destination plate.

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

**[Cavro 3]{.underline}**

NOTE: This should be performed on the first plate of each batch
dispensed during **step 3.6c**.

1.  Place the first FW sample plate on the weighing balance and press
    the **TARE** button.

2.  Place the first RV sample plate on the weighing balance and note the
    difference with the FW plate.

3.  Place both plates onto the appropriate Cavro stage.

4.  Click on **START**.

    a.  Watch for droplets on the tips and missed wells on the plate.

    b.  Visually check the bottom of the plate to make sure that the
        volumes in all of the wells are uniform.

> ***NOTE:** If tips are clogged, wells are missing, or volumes are not
> dispensed correctly, submit a work request & call **Instrumentation**
> to get the problem fixed.*

5.  Upon completion of dispensing, reweigh both plates to check the
    volume of sequencing chemistry cocktail that is being dispensed and
    record the data in the Cavro Excel spreadsheet.

6.  If weights are all within the range continue to dispense all the
    plates in the batch. Acceptable dispense range for 4ul of BD v3.1
    (0.5rxn) chemistry is **1.501-1.571g**.

> ***NOTE 1:** 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 cavro 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 2:** 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, submit a work request
> to instrumentation for calibration.*

# Procedure

NOTE 1: Make sure to wear gloves throughout the entire Sequencing
Chemistry process.

NOTE 2: Heat Inactivation (**Step 8** of the **Rolling Circle
Amplification on Plasmid Glycerol Stock** protocol) must be completed
prior to aliquoting the sequencing template into the FW and RV plates.

NOTE 3: If you are resequencing plates, go to the Venonat database 
click on **RCA DB**  click on **Scan in RCA plates for Re-Sequencing
only!**  enter in: *operator* and *direction*  click on **SUBMIT** 
print the barcodes and affix to 384-well PCR plates.

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

    1.  Remove the BigDye v3.1 Terminator Kit, respective FW/RV primers,
        & any leftover cocktail corresponding with respective primers
        from the -20°C freezer #202 located in Rm 140.

    2.  Let the reagents thaw on ice.

> ***NOTE 1:** Only remove the one primer type at a time. Return it back
> to the freezer as soon as the mix is made. Label the primer tube with
> the date and the batch number prior to replacing in the freezer. The
> ice bucket may only contain one primer type at any time.*
>
> ***NOTE 2:** DO NOT CONSOLIDATE PRIMER TUBES.*
>
> ***NOTE 3:** The BigDye Kit usually takes 30 minutes to thaw, but may
> take up to two hours.*

3.  Remove the 5X buffer from the 4^o^C deli located in Rm140 and place
    it in the same ice bucket as BigDye reagents.

4.  Place a 175 ml Corning Conical tube of fresh Milli-Q water and place
    it on ice.

> ***NOTE:** Make sure water is chilled prior to making the sequencing
> chemistry cocktail*

2.  **[Aliquoting (Hydra Twister Robot)]{.underline}**

> ***NOTE:** Make sure to randomize aliquoting batches with at least 2
> or more RCA batches that have the same primer association.*

1.  At the completion of Heat Inactivation (**Step 8** of the **Rolling
    Circle Amplification on Plasmid Glycerol Stock** protocol) FW and RV
    destination barcodes were printed. Affix these FW and RV barcodes to
    new Axygen plates (on the numbered and indented side of the plate).

> ***NOTE:** Make sure that the barcodes on the destination plates are
> marked with the correct primer association color against the database
> and against the tubes you pulled (key located on the countertop).*
>
> 2.2 Obtain the RCA source plates from final heat off of the
> thermocyclers or from the 4^o^C deli in the Sequencing Preparation
> room (Rm140) and remove the foil seals.
>
> ***Checkpoint**: Visually check the RCA source plate after removing
> the seal to ensure that the proper volume is in each plate.*
>
> 2.3 Match the source plates with their corresponding destination
> plates (the stacks of plates should be in the same order).
>
> 2.4 In the Venonat database click on **RCA DB**  **STEP 4A - Scan RCA
> Plates to Update Aliquot Information**  enter in:

a.  Select the correct information from the drop-down menus: *Hydra,
    Aliquot operator*.

b.  Use the barcode scanner to scan in the RCA source plates.

c.  Click on **SUBMIT**.

d.  Scan in the corresponding FW and RV plates and click on **SUBMIT**.

> ***Checkpoint:** If there are any mismatched plates correct the
> mismatches before loading these plates onto the Hydra Twister
> stackers.*
>
> 2.5 Place the RCA templates (source plates) in Stacker 1 and the
> destination plates (FW and RV pairs) in Stackers 3 and 4 (load stacker
> 4 up with a maximum of 32 plates before loading stacker 3). The top
> source plate should match the top destination plate in stacker 4. To
> distinguish Twister 2 plates from Twister 1 plates, add black dots to
> the FW and RV barcodes of Twister 2 plates.
>
> ***NOTE**: Press down the emergency stop button located next to the
> Hydra Twister to manually move the Twister arm.*
>
> 2.6 At the beginning of each week, move the *supply* and *drain*
> Masterflex tubing to a new slot and write the date on the tubing.
>
> ***NOTE:** There are 4 slots on the tubing. When all slots have been
> filled once, submit a work request to have the tubing changed (approx.
> every month).*
>
> 2.7 At the beginning of each day, turn on the Hydra Twister computers,
> log on, then push down and twist up each emergency stop button located
> next to the Hydra Twister.
>
> 2.8 Open the **Aliquot AUTOSTART1** program on the computer monitor.

9.  Run the **WASH_100μlx3** program.

> 2.10 After completion of the **WASH_100μlx3** program, exit the
> program.
>
> ***NOTE:** Each time a task is completed in the Aliquot Autostart1
> program, you must close the program and re-open it.*
>
> 2.11 Re-open **Aliquot AUTOSTART1** and click on **Aliquot Axygen
> 384's**.
>
> ***Checkpoint:** The program will ask if there is enough water. Check
> the supply container and if it is less than half full, press the
> **START** button located above the supply container. If the water
> container is greater than half full, click **OK** on the program
> window. This must be done for every batch.*
>
> ***NOTE 1:** This program will aliquot **1μl** per well from the RCA
> plates into each well of the corresponding FW and RV plates in
> preparation for sequencing.*
>
> ***NOTE 2:** Periodically monitor the water trough to ensure water is
> filling and draining correctly. If water trough is not filling or
> draining properly or if the program was stopped abruptly, then
> manually fill or drain the trough by pushing the corresponding button
> towards manual on the Twister Hydra Pump Controller located next to
> the Hydra Twister. Make sure to switch the button back to automatic
> when finished. If the water trough is not filling or draining
> properly, also submit a work request.*
>
> 2.12 Weigh one FW and one RV plate from each Hydra Twister (Refer to
> **Hydra Twisters** under the **Calibration Check** section).
>
> 2.13 After the program is complete and the pump has stopped running,
> close the window and push down and twist up the emergency stop button.
>
> ***NOTE:** This will move the robotic arm so that the RCA source
> plates may be removed.*
>
> 2.14 Remove source and destination plates from the Hydra-Twister
> stackers.
>
> 2.15 Re-open **Aliquot AUTOSTART1** and run the **WASH_100μlx3**
> program.
>
> ***NOTE:** This program must be run in between each batch/run.*
>
> 2.16 For the source RCA plates:

a.  Place clear adhesive seals on all source RCA plates.

b.  From each RCA batch:

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i.  Isolate 6 random plates.

    ii. Wrap the plates with labeling tape.

    iii. Label: *RCA batch \#* and *RCA date*.

    iv. Put the plates in the 4^o^C deli located in Rm 140 in the *QC
        Gels* bin.

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c.  Wrap the remaining plates in each batch with labeling tape.

d.  Label: *ABI sequencing batch \#* and *date*.

e.  Put the plates in the 4^o^C deli located in Rm 140 on the correct
    shelf for that day.

> ***NOTE:** Steps 2.16 a-e can be done while running the Cavro during
> step 3.6. After the plates have been stored in the 4^o^C deli for 1
> week, throw them away in the autoclave waste container.*
>
> 2.17 For the FW and RV plates:
>
> a\. Stack FW and RV plates into FW and RV sets.
>
> b\. Visually check destination plates to make sure that all 384 wells
> contain RCA template.
>
> ***Checkpoint:** If 4 or more wells per plate are empty or overfilled,
> re-aliquot the plate manually with a Hydra. If there is something
> wrong with a tip (e.g. clogged tip), submit a work request. If the
> source plate is the cause of the missing wells, see the supervisor to
> make a determination on how to proceed.*
>
> c\. Place a clear adhesive seal on the top plate of each stack.
>
> d\. Store the stacks in the 4^o^C deli located in Rm 140 until it is
> time to add the cocktail. If the plates are aliquoted one hour or more
> before the cocktail will be added, add a clear adhesive seal to each
> plate in the stack before storing the plates in the deli.
>
> 2.18 At the end of the day, place a trough with Coulter Clenz cleaning
> reagent (located under the sink in room 140) in the destination area
> and run the **Trough Wash** program.

a.  Enter *needle fill amount*: **80**%. Click **OK**.

b.  Enter the *number of wash cycles*: **5**. Click **OK**.

c.  Enter the *soak time*: **60** sec. Click **OK**.

d.  Enter the *number of bath wash cycles*: **5***.* Click **OK**.

> 2.19 To shut down the Hydra Twister Robot for overnight storage:

a.  Click on **End of the Day Fill** to fill the needles with water.

b.  Turn off the robot by pushing down on the red emergency stop button
    located next to the Hydra Twister.

c.  Log off of the computers.

######### Sequencing Chemistry 

1.  Label two 175-ml Corning conical tubes *FW* or *RV*, and place them
    on ice.

2.  Prepare the BigDye cocktail (Refer to the **Reagent/Stock
    Preparation** section) using the two 175-ml tubes (one for FW and
    one for RV).

> **NOTE 1:** When dispensing on Cavro 3 -- make enough cocktail for 6
> extra plates of FW and RV so the syringes don't run dry. When
> dispensing by hand -- make 20% extra cocktail for each.

3.  Cap each FW and RV BigDye cocktail tube, and mix by swirling gently
    and inverting each tube 3 to 4 times.

4.  Place two 175ml Corning conical Tubes into the respective two 600-ml
    beakers of ice labeled *FW* and *RV*.

5.  Using Cavro 3, dispense **4μl** of the FW and RV BigDye cocktail
    into each well of corresponding FW and RV 384-well plates.

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a.  Log onto the computer for Cavro 3.

b.  Click on the **Cavro 3 Barcodes** icon located on the desktop.

c.  Check the Cavro 3 calibration for each batch (Refer to the
    **Calibration Check** section).

d.  Remove the FW Cavro tubing from the FW water bottle.

e.  Replace the FW water bottle located on the FW Cavro stand with the
    FW beaker containing FW cocktail.

f.  Place the FW tubing into the FW 175 ml corning conical tube.

g.  Make sure the FW stopper is at the bottom of the tube.

h.  Repeat steps **3.6.d** -- **3.6.g** for the RV Cavro and RV
    cocktail.

i.  Under *Select Stations*, select **BOTH.**

j.  Under *Select Process*, select **PRIME**.

> ***NOTE 1:** Before priming, make sure that the troughs do not have
> water in them.*
>
> ***NOTE 2:** Ensure that the feed tubes are submersed in BigDye
> cocktail and contain no air bubbles*

k.  Dump the FW and RV trough contents back into corresponding FW and RV
    conical tubes of BigDye cocktail.

l.  Remove the FW and RV plate stacks from the 4^o^C deli located in
    Rm140.

m.  Place a FW plate onto its corresponding FW plate stand (bottom Cavro
    stage) and place a RV plate onto its corresponding RV plate stand
    (top Cavro stage).

n.  Click on **START**.

o.  Upon completion of the dispensing run, take the plates off of the
    stands and visually check to make sure that cocktail has been
    dispensed into each well.

> ***Checkpoint:** If 4 wells or less per plate are empty, manually add
> 4μl of cocktail to the empty wells with a pipette. If more than 4
> wells are empty or overfilled, re-aliquot the plate with the hydra and
> run it through the Cavro again. If there is something wrong with a tip
> (e.g. clogged tip), submit a work request and contact
> **Instrumentation** to resolve the problem.*
>
> ***NOTE:** While Cavro is dispensing, the source plates can be
> sealed.*

p.  Place the plates on ice.

q.  Repeat steps **3.6m** -- **3.6p** until cocktail has been dispensed
    into all of the plates.

> ***NOTE:** Instead of clicking on **START** in step **3.6n,** you may
> now press **ENTER** on the keyboard to start a dispensing run.*

6.  Save the leftover BigDye cocktail after each batch:

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a.  Under *Select Process*, select **PRIME**.

b.  Dump the remaining cocktail from the FW and RV 175 ml Corning
    conical tubes and FW and RV troughs into corresponding FW and RV
    50-ml tubes labeled with: *the date, either FW or RV, batch number,
    primer type, primer lot number, BigDye lot #, & 5X Buffer Lot #.*

c.  Place the tubes in the -20^o^C freezer located in Rm 140 or keep on
    ice if they will be used again.

d.  DO NOT USE leftover cocktail from a previous batch during the day.
    This should be refrozen after completing the batch.

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

8.  Use the PlateLoc Thermal Plate Sealer to seal each plate with a
    white foil seal.

9.  In the Venonat database, click on **RCA DB**  **STEP 4B - Scan
    Plates for Cycle Sequencing**:

    a.  Fill in the boxes: *Dispense Operator, Dispense Instrument, FW
        Primer Lot, FW Kit Lot, RV Primer Lot, RV Kit Lot, Mix Operator,
        Days*.

    b.  Scan in the plates in FW and RV pairs to thermocyclers according
        to which side of the thermocycler a plate will be loaded.

    c.  Click on **SUBMIT**.

    d.  Print this page and add it to the sequencing chemistry binder.

10. Place the plates on the correctly matched thermocyclers.

11. Run the **BigDye** program on the thermocyclers (refer to
    **Instrument Maintenance & Settings** for cycle program).

> ***NOTE:** Use the computer to start thermocyclers if all
> thermocyclers are filled on the corresponding rack. Otherwise, start
> thermocyclers individually. After starting thermocyclers, verify that
> each thermocycler has been started on the correct program.*

12. Wash Cavro 3 after each batch.

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a.  Place the 175ml Corning conical tubes in the autoclave bin.

b.  Place the 250-ml water bottles back on their respective FW and RV
    stands.

c.  Place the Cavro tubing back into the bottles.

d.  Under *Select Process*, select **PRIME** and repeat 3 times.

> ***NOTE:** Empty the FW and RV troughs into waste containers after
> each wash.*

e.  Rinse out the FW and RV troughs by running them under Milli-Q water.

f.  Add fresh Milli-Q water to each 250-ml bottle when the water levels
    get low.

g.  Place the FW and RV troughs back into their slots.

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13. After the thermocycler program ends (approx. 2.5 hours later),
    remove the paired plates from the thermocyclers and place them in a
    bin labeled: *ABI batch \# and date*.

> ***NOTE**: Stop the thermocyclers either manually or by computer. If
> the plates do not come out easily, use another plate or a plate
> release tool to pry the plates up off the thermocyclers. When the
> plates start getting difficult to remove, spray the thermocyclers with
> **PTFE Release Agent Dry Lubricant**.*

14. Place the plates in the 4^o^C fridge located in Rm141 for the BET
    cleanup protocol.

15. At the end of the day:

> a\. On Mondays, clean the Cavro by priming 70% Ethanol through the FW
> & RV tubes 3 times. Follow the ethanol wash with a water wash by
> priming Milli-Q water through the tubes 5 times.

b.  Replace the two 250-ml water bottles on the FW and RV Cavro stands
    with clean bottles filled with fresh Milli-Q water.

c.  Under *Select Process*, select **PRIME.**

d.  Leave the Cavro tubing filled with water.

e.  Empty & rinse out the FW and RV troughs by running them under
    Milli-Q water.

f.  Close the **Cavro 3 Barcodes** program.

g.  Log off of the computer

# Reagent/Stock Preparation

**[BigDye v3.1 Cocktail, 0.5BD reaction for Plasmid
Template]{.underline}**

[Per one 384-well Plate:]{.underline}

1.05 ml Milli-Q Water

190 μl BigDye v3.1

290 μl 5X Sequencing Buffer

6.1 μl Primer (250μM)

1.  When preparing fresh cocktail mix, prepare enough cocktail for the
    number of source plates in your sample plus 6 additional plates
    according to the *cocktail volume per number of plates* chart
    located in Sequencing Chemistry bay and also at the end of this
    protocol in **Appendix B**. To calculate how much to prepare when
    using recycled BigDye: Take the total volume you need and subtract
    the volume of leftover BigDye cocktail. Prepare cocktail based on
    the final volume.

> ***Example:** 20-source plate batch.*

a.  *You need enough cocktail volume for 26 plates/FW primer and 26
    plates/RV primer (total volume per primer = 39.896ml).*

b.  *You have 5ml leftover BigDye cocktail.*

c.  *You need to make 34.896ml fresh BigDye cocktail mix.*

d.  *Prepare enough for a 23-source plate batch (total volume per primer
    = 35.293ml) and add to the leftover BigDye cocktail.*

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2.  Place two 175 ml Corning conical tubes in the bucket. Mark each tube
    with FW or RV for the two different primer directions.

3.  Add buffer, water, BigDye, primer, and any leftover cocktail to each
    250-ml bottle and mix by swirling and inverting the bottle.

> ***NOTE 1:*** *Keep all reagents on ice while preparing cocktail and
> dispensing into the 384-well plates.*
>
> ***NOTE 2:** If a tube of leftover cocktail has not been used within
> the last month, throw it out and make completely fresh mix.*

**[70% Ethanol]{.underline}**

1.  Pour **350 ml** of 100% ethanol into a 500-ml bottle.

2.  Add **150 ml** of Milli-Q water.

3.  Mix well by inverting bottle.

4.  Label bottle: *70% ethanol, date prepared, and your initials.*

# Instrument Maintenance & Settings

**[Perkin Elmer 9700/Applied Biosystems 9700]{.underline}**

**Program Big Dye**

> **95^o^C for 1 minute**
>
> **95^o^C for 30 seconds**
>
> **50^o^C for 20 seconds 25 cycles**
>
> **60^o^C for 4 minutes**
>
> **Hold at 4^o^C**

Volume = 5μl

**[Cavro 3]{.underline}**

**Setup:**

1.  Log onto the computers located next to the Cavros.

2.  Double-click on the **Cavro 3 Barcodes** icon.

**Calibration:**

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

**Shut-down:**

1.  Close the **Cavro 3 Barcodes** program.

> ***NOTE**: Be sure that the tubes are full of water and that the water
> bottles are replaced daily with clean glassware and Milli-Q water*.

2.  Log off the computer.

# Troubleshooting

**[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**                                           
  ----------------------- ----------------------- -----------------------

# Appendix A

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

**5/27/04** -- Duane Kubischta and Bill Feil performed an audit between
6:00AM -- 12:00PM. The operator was David Robinson.

# Appendix B

  ------------ --------- ---------- ------------ ---------- ----------- -----------
                                    **ABI                               
                                    Sequencing                          
                                    Chemistry-                          
                                    Reduced                             
                                    Buffer (0.5                         
                                    BD rxn)**                           

  When                                                                  
  dispensing                                                            
  with *Cavaro                                                          
  3*, prepare                                                           
  mix for *6                                                            
  more plates*                                                          
  than the                                                              
  number of                                                             
  plates in                                                             
  the batch.                                                            

                                                                        

  **Plate \#** **Water   **BigDye   **5X Buffer  **Primer   **Total in  
               (ml)**    (ml)**     (ml)**       (ul)**     ea. Mix     
                                                            (ml)**      

  1            1.05      0.19       0.29         6.1        1.534       

  2            2.10      0.38       0.58         12.3       3.069       

  3            3.14      0.58       0.86         18.4       4.603       

  4            4.19      0.77       1.15         24.6       6.138       

  5            5.24      0.96       1.44         30.7       7.672       

  6            6.29      1.15       1.73         36.9       9.207       

  7            7.34      1.34       2.02         43.0       10.741      

  8            8.39      1.54       2.30         49.2       12.276      

  9            9.43      1.73       2.59         55.3       13.810      

  10           10.48     1.92       2.88         61.4       15.345      

  11           11.53     2.11       3.17         67.6       16.879      

  12           12.58     2.30       3.46         73.7       18.414      

  13           13.63     2.50       3.74         79.9       19.948      

  14           14.68     2.69       4.03         86.0       21.482      

  15           15.72     2.88       4.32         92.2       23.017      

  16           16.77     3.07       4.61         98.3       24.551      

  17           17.82     3.26       4.90         104.4      26.086      

  18           18.87     3.46       5.18         110.6      27.620      

  19           19.92     3.65       5.47         116.7      29.155      

  20           20.97     3.84       5.76         122.9      30.689      

  21           22.01     4.03       6.05         129.0      32.224      

  22           23.06     4.22       6.34         135.2      33.758      

  23           24.11     4.42       6.62         141.3      35.293      

  24           25.16     4.61       6.91         147.5      36.827      

  25           26.21     4.80       7.20         153.6      38.362      

  26           27.26     4.99       7.49         159.7      39.896      

  27           28.30     5.18       7.78         165.9      41.431      

  28           29.35     5.38       8.06         172.0      42.965      

  29           30.40     5.57       8.35         178.2      44.499      

  30           31.45     5.76       8.64         184.3      46.034      

  31           32.50     5.95       8.93         190.5      47.568      

  32           33.55     6.14       9.22         196.6      49.103      

  33           34.59     6.34       9.50         202.8      50.637      

  34           35.64     6.53       9.79         208.9      52.172      

  35           36.69     6.72       10.08        215.0      53.706      

  36           37.74     6.91       10.37        221.2      55.241      

  37           38.79     7.10       10.66        227.3      56.775      

  38           39.84     7.30       10.94        233.5      58.310      

  39           40.88     7.49       11.23        239.6      59.844      

  40           41.93     7.68       11.52        245.8      61.379      

  41           42.98     7.87       11.81        251.9      62.913      

  42           44.03     8.06       12.10        258.0      64.447      

  43           45.08     8.26       12.38        264.2      65.982      

  44           46.13     8.45       12.67        270.3      67.516      

  45           47.17     8.64       12.96        276.5      69.051      

  46           48.22     8.83       13.25        282.6      70.585      

  47           49.27     9.02       13.54        288.8      72.120      

  48           50.32     9.22       13.82        294.9      73.654      

  49           51.37     9.41       14.11        301.1      75.189      

  50           52.42     9.60       14.40        307.2      76.723      

  51           53.46     9.79       14.69        313.3      78.258      

  52           54.51     9.98       14.98        319.5      79.792      

  53           55.56     10.18      15.26        325.6      81.327      

  54           56.61     10.37      15.55        331.8      82.861      

  55           57.66     10.56      15.84        337.9      84.396      

  56           58.71     10.75      16.13        344.1      85.930      

  57           59.75     10.94      16.42        350.2      87.464      

  58           60.80     11.14      16.70        356.4      88.999      

  59           61.85     11.33      16.99        362.5      90.533      

  60           62.90     11.52      17.28        368.6      92.068      

  61           63.95     11.71      17.57        374.8      93.602      

  62           65.00     11.90      17.86        380.9      95.137      

  63           66.04     12.10      18.14        387.1      96.671      

  64           67.09     12.29      18.43        393.2      98.206      

  65           68.14     12.48      18.72        399.4      99.740      

  66           69.19     12.67      19.01        405.5      101.275     

  67           70.24     12.86      19.30        411.6      102.809     

  68           71.29     13.06      19.58        417.8      104.344     

  69           72.33     13.25      19.87        423.9      105.878     

  70           73.38     13.44      20.16        430.1      107.412     
  ------------ --------- ---------- ------------ ---------- ----------- -----------

#