Cycle Sequencing using BigDye v3.1

**Performed on Rolling Circle Amplified DNA Template**

1/8^th^ Reaction (1.0BD)

> 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: Nahal Lalefar

# 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 |
| *[Disposables]{.underline}*     | Axygen/ISC BioExpress | derline}** |
|                                 |                       |            |
| 384-well PCR Microplate, Clear  | Falcon/VWR            | T-3059-1CS |
|                                 |                       |            |
| 125-ml Conical Tube             | Corning/VWR           | \-         |
|                                 |                       |            |
| 50-ml Centrifuge Tube           | Velocity 11           | 430290     |
|                                 |                       |            |
| PlateLoc Peelable Heat Seal     | Rainin                | 06643-001  |
|                                 |                       |            |
| LTS Pipette Tips (2, 10, 100,   | Applied Biosystems    | \-         |
| 200, 1000μl)                    |                       |            |
|                                 | Applied Biosystems    | 4337458    |
| *[Reagents]{.underline}*        |                       |            |
|                                 | IDT                   | 4336701    |
| BigDye Terminator v3.1 Cycle    |                       |            |
| Sequencing Kit                  | IDT                   | \-         |
|                                 |                       |            |
| 5X BigDye Terminator v1.1/v3.1  | Beckman Coulter       | \-         |
| Sequencing Buffer               |                       |            |
|                                 | AAPER Alcohol (LBNL)  | PN 8546930 |
| FW Sequencing Primer            |                       |            |
|                                 | Millipore Milli-Q     | 6810-13289 |
| RV Sequencing Primer            | System                |            |
|                                 |                       | \-         |
| Coulter Clenz                   | Robbins Scientific    |            |
|                                 |                       | \-         |
| 100% Ethanol (200 Proof)        | Robbins Scientific    |            |
|                                 |                       | \-         |
| Milli-Q Water                   | JGI                   |            |
|                                 |                       | \-         |
| *[Equipment]{.underline}*       | Rainin                |            |
|                                 |                       | \-         |
| 384-well Hydra Twister          | Perkin Elmer (Applied |            |
|                                 | Biosystems)           | \-         |
| Hydra Wash Reservoir            |                       |            |
|                                 | Velocity 11           | \-         |
| Cavro Dispensing Robot          |                       |            |
|                                 |                       |            |
| Pipettes (2, 10, 100, 200,      |                       |            |
| 1000μl)                         |                       |            |
|                                 |                       |            |
| GeneAmp PCR System 9700         |                       |            |
|                                 |                       |            |
| PlateLoc Thermal Plate Sealer   |                       |            |
+---------------------------------+-----------------------+------------+

# Calibration Check

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

This should be performed prior to the dispensing run for each batch at
Step **2.9.c**.

1.  Make sure that the FW and RV tubing are in their respective Milli-Q
    water bottles.

2.  On the desktop located next to Cavro, double-click on the **Cavro 3
    Barcodes** icon.

> ***NOTE:** Make sure only one program is open at a time.*

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

4.  Under *Select Process*, select **PRIME** (Ensure that the feed tubes
    contain no air bubbles).

5.  Place an empty 384-well plate on the weighing balance and press the
    **TARE** button.

6.  Place the tared empty plate onto the *bottom* Cavro stage.

7.  Under *Select Stations*, select the **BOTTOM** station.

8.  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.

9.  Upon completion of dispensing, reweigh the plate to check the volume
    of water that is being dispensed and record the data in the Cavro
    notebook.

> ***NOTE**: If the weight ranges fall outside of **1.49g -- 1.58g**,
> submit a work request for calibration.*

10. Repeat steps **4** -- **5**.

11. Place the tared empty plate into the *top* Cavro stage.

12. Under *Select Stations*, select the **TOP** station.

13. Repeat steps **8** -- **9**.

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

15. Under *Select Process*, select **RECYCLE**.

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

This should be performed on one FW and one RV plate within the first 10
plates per batch per Hydra Twister at step **1.11**.

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**: If the weight ranges fall outside of **0.3072g --
> 0.4608g**, submit a work request for calibration.*

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

> ***NOTE:** To open excel spreadsheet click on **Shortcut to Plate
> Weight** 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 Hydra Twister Calibration click on
> Plate WeightData.*

7.  Using the same aliquoted, weighed FW destination plate, measure the
    volume of a single well using the 2l pipette.

> ***NOTE 1:** If an accurate reading cannot be taken on the well,
> measure the volume of another well until an accurate reading can be
> taken. It may take up to 3 or 4 wells for an accurate reading.*
>
> ***NOTE 2:** FW plates have a volume of approximately **1.2μl**. RV
> plates have a volume of approximately **1.3-1.35μl**.*

8.  Enter the volume into the Plate Weight Excel Spreadsheet.

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

10. Repeat steps **1 -- 9** with a RV destination plate.

# Procedure

NOTE 1: Heat Inactivation (**Step 5** of the **Rolling Circle
Amplification on Plasmid Glycerol Stock** protocols) must be completed
prior to aliquoting the sequencing template into the FW and RV plates.

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

> ***NOTE:** Make sure that the barcodes on the destination plates are
> marked with the correct primer association color.*

1.  Obtain the RCA source plates from the 4^o^C deli in the Sequencing
    Preparation room (Rm140) and remove the foil seals.

2.  Match the source plates with the corresponding destination plates
    (i.e. The stacks of plates should be in the same order).

3.  In the Venonat database, click on **RCA DB** **STEP 4A - Scan RCA
    Plates to Update Aliquot Information** enter in:

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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.*

4.  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).
    To distinguish Twister 2 plates from Twister 1 plates, add black
    dots to the FW and RV barcodes of Twister 2 plates.

5.  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 twice, submit a work request to have the tubing changed
> (approx. every 2 months).*

6.  At the beginning of each day, open the Hydra Twister laptop
    monitors, log on, then push down and twist up each emergency stop
    button.

7.  Open the **Aliquot Autostart** program on the laptop monitor.

8.  Run the **WASH_100μlx3** program.

9.  After completion of the **WASH_100μlx3** program, exit the program.

> ***NOTE:** Each time a task is completed in the Aliquot Autostart
> program, you must shut the program down and re-open it.*

10. Re-open **Aliquot Autostart** 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 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.*

11. Weigh one FW and one RV plate from each Hydra Twister (Refer to
    **Hydra Twisters** under the **Calibration Check** section).

12. After the program is complete and the pump has stopped running,
    close the window and push down and twist up the emergency stop
    button. This will move the robotic arm so that the RCA source plates
    may be removed.

13. Run the **WASH_100μlx3** program in between each batch/run.

14. Visually check 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.*

15. For the source RCA plates:

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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 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 on the correct shelf for that day.

    4.  For the FW and RV plates:

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a.  Stack FW and RV plates into FW and RV sets.

b.  Place a clear adhesive seal on the top plate of each stack.

c.  Store the stacks in a 4^o^C deli 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.

    4.  At the end of the day, place a trough with Coulter Clenz
        cleaning reagent in the destination area and run the **Trough
        Wash** program.

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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**.

    4.  To shut down the Hydra Twister Robot for overnight storage:

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

b.  Turn off the robot.

c.  Log off of the laptop.

######### 2. Sequencing Chemistry 

1.  Remove the BigDye v3.1 Terminator Kit, and FW/RV primers from the
    -20C freezer.

2.  Remove the 5X buffer from the 4^o^C deli.

3.  Let the reagents thaw on ice.

> ***NOTE:** The BigDye Kit usually takes 30 minutes to thaw but may
> take up to two hours*.

4.  Label two 250-ml bottles *FW* or *RV*, respectively, and place them
    on ice.

5.  Prepare the BigDye cocktail (Refer to the **Reagent/Stock
    Preparation** section) using the two 250-ml bottles (one bottle for
    FW and one bottle for RV).

> **NOTE:** 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.

6.  Cap each FW and RV BigDye cocktail bottle, and mix by swirling
    gently and inverting each bottle 3 times.

7.  Place two 125-ml conical tubes into two 600-ml beakers of ice
    labeled *FW* and *RV*.

8.  Pour the FW cocktail into the 125-ml conical tube located in the FW
    beaker and pour the RV cocktail into the 125-ml conical tube located
    in the RV beaker.

9.  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 conical tube.

g.  Make sure the FW stopper is at the bottom of the conical tube. If it
    is not, tape the glass rod of the Cavro tubing to the conical tube.

h.  Repeat steps **2.9.d** -- **2.9.g** for the RV Cavro and RV
    cocktail.

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

> ***NOTE:** Before priming, make sure that the troughs do not have
> water in them.*

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

k.  Remove the FW and RV plate stacks from the 4^o^C deli.

l.  Place the FW or RV plate onto the corresponding FW or RV plate
    stand.

m.  Click on **START**.

n.  Upon completion of the dispensing run, take the plates off of the
    stands and visually check to make sure that the 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.*

o.  Place the plates on ice.

p.  Add the next set of FW and RV plates to the FW or RV plate stand.

q.  Click on the **ENTER** key on the keyboard.

r.  Repeat steps **2.9.n** -- **2.9.q**. until cocktail has been
    dispensed into all of the plates.

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9.  Save the leftover BigDye cocktail after each batch:

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

    b.  Dump the remaining cocktail from FW and RV 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, and
        primer lot number.*

    c.  Place the tubes in the -20^o^C freezer.

10. Wash Cavro 3 after each batch.

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

    b.  Place the Cavro tubing back into the bottles.

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

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

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

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

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

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

12. Use the PlateLoc Thermal Plate Sealer to seal the plates with foil
    seals.

13. 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 according to which side of
        the thermocycler it will be loaded.

    c.  Click on **SUBMIT**.

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

14. Place the plates on the correctly matched thermocyclers.

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

16. After the program ends, remove the paired plates from the
    thermocyclers and place them in a bin labeled: *ABI batch \# and
    date*.

17. Place the plates in the 4^o^C fridge for the BET cleanup protocol.

18. 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.  Leave the Cavro tubing filled with water.

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

    e.  Log off of the computer.

# Reagent/Stock Preparation

**[BigDye v3.1 Cocktail, 1.0BD reaction for Plasmid Template (per one
384-well plate)]{.underline}**

1.0 ml Milli-Q Water

400 μl BigDye version 3.1

200 μl 5X Sequencing Buffer

6.1 μl Primer (250μM)

1.  When preparing a 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. To calculate how much to
    prepare: 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.  Add buffer, water, BigDye, primer, and any leftover cocktail to a
    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 a 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.

2.  Log off the computer.