Cycle Sequencing using BigDye v3.1

**Performed on Fosmid DNA Template**

> Version Number: 1.3
>
> Production Start Date: 02/01/04
>
> Version 1.3 Date: 06/11/04
>
> Authors: Arshi Khan, Jamie Jett
>
> Reviewed/Revised by: Catherine Gordon, Megha Ambati

# Summary

The BigDye v3.1 sequencing kit is used to cycle sequence fosmid DNA,
prepared via Agencourt SprintPrep or CosMCPrep, in preparation for
sequencing on the ABI 3730xl instruments.

# Materials & Reagents

+--------------------------------+----------------------+--------------+
| **[Materials/Rea               | **[V                 | **[Stock     |
| gents/Equipment]{.underline}** | endor]{.underline}** | Number]{.    |
|                                |                      | underline}** |
| *[Disposables]{.underline}*    | Axygen/ISC           |              |
|                                | BioExpress           | T-3059-1CS   |
| 384-well PCR Microplate, Clear |                      |              |
|                                | Corning/VWR          | 2533-50      |
| 50-ml Centrifuge Tube          |                      |              |
|                                | Falcon/VWR           | 357525       |
| 25-ml Serological Pipet        |                      |              |
|                                | Falcon/VWR           | 357551       |
| 10-ml Serological Pipet        |                      |              |
|                                | Velocity 11          | 06643-001    |
| PlateLoc Peelable Heat Seal    |                      |              |
|                                | Rainin               | \-           |
| LTS Pipette Tips (2, 10, 100,  |                      |              |
| 200, 1000μl)                   | Matrix               | 8086         |
|                                |                      |              |
| 100-ml Reagent Reservoir       | Applied Biosystems   | 4337458      |
|                                |                      |              |
| *[Reagents]{.underline}*       | Applied Biosystems   | 4336701      |
|                                |                      |              |
| BigDye Terminator v3.1 Cycle   | IDT                  | \-           |
| Sequencing Kit                 |                      |              |
|                                | IDT                  | \-           |
| 5X BigDye Terminator v1.1/v3.1 |                      |              |
| Sequencing Buffer              | AAPER Alcohol (LBNL) | 6810-13289   |
|                                |                      |              |
| T7 Fosmid FW Sequencing Primer | Millipore Milli-Q    | \-           |
| (250μm)                        | System               |              |
|                                |                      | \-           |
| *5' TAA TAC GAC TCA CTA TAG GG | \-                   |              |
| 3'*                            |                      | \-           |
|                                | Thermo electron      |              |
| T7 Fosmid RV Sequencing Primer |                      | \-           |
| (250μm)                        | Thermo electron      |              |
|                                |                      | \-           |
| *5' ATG ACC ATG ATT ACG CCA AG | Robbins Scientific   |              |
| 3'*                            |                      | \-           |
|                                | Robbins Scientific   |              |
| 100% Ethanol (200 Proof)       |                      | \-           |
|                                | Perkin Elmer         |              |
| Milli-Q Water                  | (Applied Biosystems) | \-           |
|                                |                      |              |
| Coulter Clenz                  | Velocity 11          | \-           |
|                                |                      |              |
| *[Equipment]{.underline}*      | Eppendorf            | \-           |
|                                |                      |              |
| 96/384-well Multidrop Micro    | Eppendorf            |              |
|                                |                      |              |
| Multidrop Micro Cassette Head  |                      |              |
|                                |                      |              |
| 384-well Hydra Twister         |                      |              |
|                                |                      |              |
| Hydra Wash Reservoir           |                      |              |
|                                |                      |              |
| GeneAmp PCR System 9700        |                      |              |
|                                |                      |              |
| PlateLoc Thermal Plate Sealer  |                      |              |
|                                |                      |              |
| Centrifuge 5810                |                      |              |
|                                |                      |              |
| Easypet                        |                      |              |
+--------------------------------+----------------------+--------------+

# Calibration Check

**[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 **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 **1.463g -- 1.55g**,
> WARNING.*
>
> ***NOTE 2**: If the weight ranges fall outside of **1.441g** --
> **1.572g** DO NOT USE.*

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

> ***NOTE:** To open the 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 RCAclick on Calibrations click on Hydra Twister
> Calibration click on Plate WeightData select Fosmid 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 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.

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

NOTE 1: This should be performed on the 1^st^ plate per Multidrop Micro
instrument per each Fosmid sequencing chemistry batch.

NOTE 2: Make sure the Multidrop Micro is set for a 384 plate, 2µl
volume, and 24 columns (col).

NOTE 3: Refer to the **Multidrop Micro** in the **Instrument Maintenance
& Settings** at the end of this protocol for proper Priming, Dispensing,
and Purging techniques.

1.  Prior to dispensing any liquid, go to the balance and tare an empty
    384-well Axygen plate.

2.  Dispense **2μl** of Milli-Q water into each well.

3.  Visually check to make sure that Milli-Q water 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 plates
    in batch. If not, set the cassette aside for the Instrumentation
    group and change to a new cassette; when changing to a new cassette,
    perform a calibration check on new cassette head by repeating steps
    **1** -- **4**.

> ***NOTE**: Acceptable weight range after 2μl Milli-Q water addition:
> **0** **.741g --0 .875 g**.*

6.  Record these values in the **MD Calibrations** Excel Spreadsheet,
    along with the serial number of the Multidrop Micro cassette head.

> ***NOTE 1:** To open the 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 RCAclick on MD Calibrationclick on MD
> Calibrations select Fosmid FW or Fosmid RV 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).*

# Procedure

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

NOTE 2: All reagents/stock solutions should be prepared prior to the
start of the procedure.

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 -20C freezer #202 located in Rm 140.

    2.  Let the reagents thaw on ice.

> ***NOTE 1:** 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.

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1.  **[Aliquoting the Fosmid Template]{.underline}**

    1.  Take the FW and RV fosmid barcodes located on the shelf on the
        edge of the Sequencing Chemistry bay C (placed there by
        operators in the Libraries group who processed the fosmid DNA)
        and mark a regular green stripe next to the barcodes.

    2.  Affix the barcodes to new 384-well Axygen plates (on the
        indented side of the plate), stacking the RV direction of each
        set on top of the FW direction.

    3.  Remove the fosmid template plates from the bin labeled "Fosmids
        for Sequencing" from the 4^o^C deli in the Sequencing
        Preparation Rm140.

    4.  Unseal all the fosmid source plates.

    5.  Match the fosmid source plates to the correct destination plates
        (the bottom plate in the source stack should match the bottom
        FW/RV plates of the destination stack).

    6.  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 fosmid 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 fosmid 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, log onto the Hydra Twister computers,
then push down and twist up each emergency stop button located next to
each Hydra Twister.

> 2.8 Open the **Fosmid Autostart** program on the monitor.
>
> 2.9 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 FOSMID 1
> program, close the program and re-open it.*
>
> 2.11 Re-open **Fosmid 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 **4μl** per well from the
> Fosmid source 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 fosmid source
> plates may be removed.*
>
> 2.14 Remove source and destination plates from the Hydra-Twister
> stackers.

15. Re-open **Fosmid Autostart** and run the WASH_100**μlx3** program.

> ***NOTE:** This program must be run in between each batch/run.*

16. Visually check plates to make sure that all 384 wells contain fosmid
    template.

> ***Checkpoint:** If 4 or more wells are missing or overfilled per
> plate, re-aliquot the plate with the hydra. If there is something
> wrong with the tip, submit a work request. If the source plate is the
> source of the missing wells, see a supervisor to make a determination
> on how to proceed.*
>
> 2.16 For the source fosmid plates:

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

b.  From each fosmid batch:

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i.  Isolate 1 plate per operator per library per day.

    ii. Wrap the plates with labeling tape.

    iii. Label: *Fosmids* and *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: *Fosmids* and *date*.

e.  Put the plates in the *Fosmids bin* located on the bottom shelf of
    the 4^o^C deli in Rm 140.

> 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 fosmid 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 single tip (e.g. clogged tip), submit a work request and
> have it repaired immediately. 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. ***NOTE:*** *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 computer.

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2.  **[Sequencing Chemistry]{.underline}**

    1.  Label two 50-ml Corning tubes: *FW* and *RV*, respectively, and
        place the tubes on ice to chill. Affix a 2" piece of tape to the
        side of each tube, with the *date, direction, primer type,
        primer lot#, BigDye lot#, and buffer lot#.*

    2.  Prepare the Fosmid BigDye cocktail (Refer to the **Reagent/Stock
        Preparation** section) using the two 50-ml tubes (one tube for
        FW and one for RV).

> ***NOTE 1:** When dispensing with the Multidrop Micro, prepare 30%
> extra cocktail mix for batches of 19 source plates or less and 6 extra
> plates for batches of 20 or more source plates.*
>
> ***NOTE 2:** Make fresh Fosmid BigDye cocktail at the beginning of
> every week and continue to recycle the leftovers throughout the week.*

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

4.  Install the Multidrop Micro cassette head labeled "Fosmid FW" into
    either MD Micro 1 or MD Micro 4 and install the Multidrop Micro
    cassette head labeled "Fosmid RV" into the remaining MD Micro (Refer
    to the **Installing the cassette** instructions under the
    **Multidrop Micro** section in **Instrument Maintenance &
    Settings**).

> ***NOTE:** Fosmid BigDye Cocktail can be dispensed into both FW and RV
> plates simultaneously by using the 2 different Multidrop Micro
> instruments; one for the FW direction and one for the RV direction.*

5.  Wash the Multidrop Micro tubing by priming it with Milli-Q water
    (Refer to the **Priming** instructions under the **Multidrop Micro**
    section in **Instrument Maintenance** **& Settings**).

> ***NOTE:** Make sure to fill the trough 3 times when priming with
> Milli-Q water. Every time the trough is nearly full, remember to empty
> its contents into a waste container.*

6.  Calibrate the Multidrop Micro (refer to **Mutlidrop Micro** under
    the **Calibration Check** section).

7.  Empty the water in the microtubing back into the Milli-Q water
    reservoir.

8.  Prime the Multidrop Micro containing the "Fosmid FW" cassette head
    with the FW primer BigDye cocktail and prime the Multidrop Micro
    containing the "Fosmid RV" cassette head with the RV primer BigDye
    cocktail.

> ***NOTE:** When dispensing FW cocktail, only use Multidrop Micro
> cassette head labeled as "Fosmid FW"; when dispensing RV cocktail,
> only use Multidrop Micro cassette head labeled as "Fosmid RV."*

9.  Dispense **2μl** of the cocktail into each well using the Multidrop
    Micro (Refer to the **Dispensing** instructions under the
    **Multidrop Micro** section in **Instrument Maintenance** **&
    Settings**).

> ***NOTE 1:** Monitor the 50-ml tube to ensure that there is enough
> cocktail for the plates and that the end of the Multidrop Micro tubing
> is submersed completely in the cocktail.*
>
> ***NOTE 2:*** *If a clog develops or some other Multidrop Micro
> cassette failure occurs in the middle of dispensing, stop processing
> the batch, make a list of the plates in that batch that have not had
> liquid dispensed into them, and place a comment in the database
> regarding the new Multidrop Micro cassette's unique identifying
> number. Continue processing the batch with a new cassette (after
> performing a calibration check on the new cassette). Note any changes
> in the* **MD Calibrations** *Excel Spreadsheet as well.*

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

> ***Checkpoint:** If 4 wells or less per plate are empty or low,
> manually add 2μl* *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 Multidrop Micro again. If there is
> something wrong with a tip (e.g. clogged tip), submit a work request.*

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

12. Seal the 384-well plates with the Plateloc heat sealer.

13. Purge, clean, and remove the buffer 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:** The Fosmid FW and Fosmid RV cassettes will be used daily
> until they fall out of calibration or a clog develops.*

14. Save the leftover Fosmid BigDye cocktail after each batch by placing
    the tubes in the -20^o^C freezer.

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

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

> ***NOTE:** If using different Multidrop Micro machines to dispense FW
> and RV cocktail, scan in FW and RV plates as separate batches. Select
> the appropriate Dispense Instrument when scanning plates in.*

b.  Scan in the plates according to which side of the thermocycler they
    will be loaded.

c.  Click on **SUBMIT**.

d.  Print this page and place it in the sequencing chemistry binder.

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16. Load the plates on the correctly matched thermocyclers and close the
    lids.

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

18. After the cycle sequencing is completed (approx. 6 hours), remove
    plates from thermocyclers and place them in bins labeled *ABI
    Fosmids and date run*.

19. Place the plates in the 4ºC fridge in Rm141 for the BET cleanup
    protocol.

# Reagent/Stock Preparation

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

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

8.79 μl Milli-Q Water

316 μl BigDye v3.1

424 μl 5X Sequencing Buffer

19.2 μl Primer (250μM)

1.  When preparing fresh cocktail mix for dispensing with the Multidrop
    Micro, prepare 30% extra cocktail mix for batches of 19 source
    plates or less and 6 extra plates for batches of 20 or more source
    plates according to the *cocktail volume per number of plates* chart
    located in Sequencing Chemistry bay and also in Appendix A of this
    protocol**.** To calculate how much to prepare when using recycled
    Fosmid 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 = 19.968ml).*

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

c.  *You need to make 14.968ml fresh BigDye cocktail mix.*

d.  *Prepare enough for a 20-source plate batch (total volume per primer
    = 15.360ml) and add to the leftover BigDye cocktail.*

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2.  Place two 50 ml corning tubes in a beaker filled with ice. Affix a
    2" piece of tape to the side of each tube with the *date, direction,
    primer type, primer lot#, BigDye lot#, and buffer lot#.*

3.  Add buffer, water, BigDye, primer, and any leftover cocktail to each
    50-ml corning tube and mix by inverting the tube.

> ***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 FOSMID**

**95^o^C for 1 minute**

**95^o^C for 10 seconds**

**52^o^C for 5 seconds 99 cycles**

**60^o^C for 2.5 minutes**

**Hold at 4^o^C**

Volume = 6μl

**[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 not damaged.

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 Milli-Q
> water.*
>
> ***NOTE 2**: Use separate wash bottles for FW and RV 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,
    completely hitting inside the wells).

> ***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 (trough full) followed by,
    70% ethanol (trough full), and then followed by a separate bottle of
    Milli-Q water (trough 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.

# 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

1\. BigDye v3.1 cocktail reagents per number of 384-well plates.

  ------------ --------- ---------- ------------- ------------- -----------------
  **ABI                                                         
  Sequencing                                                    
  Chemistry-                                                    
  Fosmid                                                        
  Sprint                                                        
  Prep**                                                        

  When                                                          
  dispensing                                                    
  with the                                                      
  *Multidrop                                                    
  Micro*,                                                       
  prepare 30%                                                   
  extra mix                                                     
  for batches                                                   
  of 19 source                                                  
  plates or                                                     
  less and                                                      
  prepare mix                                                   
  for 6 extra                                                   
  plates for                                                    
  batches of                                                    
  20 or more                                                    
  source                                                        
  plates.                                                       

                                                                

  **No. of     **Water   **BigDye   **5X Buffer   **Primer      **Total in ea.
  plates**     (μl)**    (ml)**     (ml)**        (μl)**        Mix (ml)**

  1            8.79      0.316      0.424         19.2          0.768

  2            17.59     0.632      0.848         38.4          1.536

  3            26.38     0.948      1.272         57.6          2.304

  4            35.17     1.264      1.696         76.8          3.072

  5            43.97     1.580      2.120         96.0          3.840

  6            52.76     1.896      2.544         115.2         4.608

  7            61.56     2.212      2.968         134.4         5.376

  8            70.35     2.528      3.392         153.6         6.144

  9            79.14     2.844      3.816         172.8         6.912

  10           87.94     3.160      4.240         192.0         7.680

  11           96.73     3.476      4.664         211.2         8.448

  12           105.52    3.792      5.088         230.4         9.216

  13           114.32    4.108      5.512         249.6         9.984

  14           123.11    4.424      5.936         268.8         10.752

  15           131.90    4.740      6.360         288.0         11.520

  16           140.70    5.056      6.784         307.2         12.288

  17           149.49    5.372      7.208         326.4         13.056

  18           158.28    5.688      7.632         345.6         13.824

  19           167.08    6.004      8.056         364.8         14.592

  20           175.87    6.320      8.480         384.0         15.360

  21           184.67    6.636      8.904         403.2         16.128

  22           193.46    6.952      9.328         422.4         16.896

  23           202.25    7.268      9.752         441.6         17.664

  24           211.05    7.584      10.176        460.8         18.432

  25           219.84    7.900      10.600        480.0         19.200

  26           228.63    8.216      11.024        499.2         19.968

  27           237.43    8.532      11.448        518.4         20.736

  28           246.22    8.848      11.872        537.6         21.504

  29           255.01    9.164      12.296        556.8         22.272

  30           263.81    9.480      12.720        576.0         23.040

  31           272.60    9.796      13.144        595.2         23.808

  32           281.40    10.112     13.568        614.4         24.576

  33           290.19    10.428     13.992        633.6         25.344

  34           298.98    10.744     14.416        652.8         26.112

  35           307.78    11.060     14.840        672.0         26.880

  36           316.57    11.376     15.264        691.2         27.648

  37           325.36    11.692     15.688        710.4         28.416

  38           334.16    12.008     16.112        729.6         29.184

  39           342.95    12.324     16.536        748.8         29.952

  **No. of     **Water   **BigDye   **5X Buffer   **Primer      **Total in ea.
  plates**     (μl)**    (ml)**     (ml)**        (μl)**        Mix (ml)**

  40           351.74    12.640     16.961        768.0         30.720

  41           360.54    12.956     17.385        787.2         31.488

  42           369.33    13.272     17.809        806.4         32.256

  43           378.12    13.588     18.233        825.6         33.024

  44           386.92    13.904     18.657        844.8         33.792

  45           395.71    14.220     19.081        864.0         34.560

  46           404.51    14.536     19.505        883.2         35.328

  47           413.30    14.852     19.929        902.4         36.096

  48           422.09    15.168     20.353        921.6         36.864

  49           430.89    15.484     20.777        940.8         37.632

  50           439.68    15.800     21.201        960.0         38.400
  ------------ --------- ---------- ------------- ------------- -----------------

# Appendix B

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

**5/27/04** -- Erika Lindquist and Sanjay Israni performed an audit
between 7:16PM -- 9:30PM. The operator was Megha Ambati.

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

**8/10/04** No longer taking individual well volumes per FW & RV plate
per batch per Hydra Twister.

**8/12/04** We started using 2 Multidrop Micros to dispense the
chemistry simultaneously; one instrument for the FW direction & one for
the RV direction.

**8/11/04** Preparing fresh Fosmid BigDye cocktail at the beginning of
every week, and recycling the leftovers throughout the week.

**9/02/04 --** Multidrop calibration weight range changed to 0.741 g-
0.875 g.

**10/20/04** -- HTS weight ranges have changed. If weights fall outside
1.463 g-- 1.55g it gets a Warning level, if it falls outside 1.441 g --
1.572g it gets a do not use level.