DYEnamic ET-Terminator Cycle Sequencing

**on Rolling Circle Amplified DNA**

10μl Automated Reaction

> Version Number: 2.1
>
> Production Start Date: 06/01/03
>
> Version 2.1 Date: 08/23/04
>
> Authors: Arshi Khan, Nancy Hammon, Catherine Gordon,
>
> Reviewed/Revised by: Amber Nivens

# Summary

The DYEnamic ET-Terminator sequencing kit is used to cycle sequence
plasmid DNA, which has been amplified by rolling circle amplification,
in preparation for sequencing on the MegaBACE 4000 instruments.

# Materials & Reagents

+------------------------------+-------------------------+------------+
| **[Materials/Reage           | *                       | **[Stock   |
| nts/Equipment]{.underline}** | *[Vendor]{.underline}** | N          |
|                              |                         | umber]{.un |
| *[Disposables]{.underline}*  | Axygen/ISC BioExpress   | derline}** |
|                              |                         |            |
| 384-well PCR Microplate,     | Corning/VWR             | T-3059-1CS |
| Clear                        |                         |            |
|                              | Velocity 11             | \-         |
| 50-ml Centrifuge Tube        |                         |            |
|                              | Rainin                  | 430290     |
| PlateLoc Peelable Heat Seal  |                         |            |
|                              | GE Healthcare\*         | 06643-001  |
| LTS Pipette Tips (2, 10,     |                         |            |
| 100, 200, 1000μl)            | IDT                     | \-         |
|                              |                         |            |
| *[Reagents]{.underline}*     | IDT                     | 93-81096   |
|                              |                         |            |
| DYEnamic ET-Terminator       | Beckman Coulter         | \-         |
| Sequencing Premix            |                         |            |
|                              | AAPER Alcohol (LBNL)    | \-         |
| FW Sequencing Primer         |                         |            |
|                              | Millipore Milli-Q       | PN 8546930 |
| RV Sequencing Primer         | System                  |            |
|                              |                         | 6810-13289 |
| 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         |                         |            |
|                              | Miller-Stephenson       | \-         |
| Cavro Dispensing Robot       |                         |            |
|                              | Velocity 11             | \-         |
| Pipettes (2, 10, 100, 200,   |                         |            |
| 1000μl)                      | Pyrex                   | MS-122V    |
|                              |                         |            |
| GeneAmp PCR System 9700      | Pyrex                   | \-         |
|                              |                         |            |
| PTFE Release Agent Dry       | Matrix                  | \-         |
| Lubricant                    |                         |            |
|                              | Falcon/VWR              | \-         |
| PlateLoc Thermal Plate       |                         |            |
| Sealer                       |                         |            |
|                              |                         |            |
| 250 ml, 500 ml bottles       |                         |            |
|                              |                         |            |
| 1000 ml beakers              |                         |            |
|                              |                         |            |
| Multi-channel 250µl Pipette  |                         |            |
|                              |                         |            |
| 250-ml Double pharmaceutical |                         |            |
| graduates                    |                         |            |
+------------------------------+-------------------------+------------+

\* GE Healthcare, formerly Amersham Biosciences

# 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 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 Calibrations click
> on Hydra Twister Calibration  click on Plate WeightData.*
>
> ***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 1]{.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.

> ***NOTE 1:** To open excel spreadsheet click on **Shortcut to Cavro
> Calibrations** on desktop. If shortcut is unavailable go to **Network
> Places** on desktop and click on **Entire Network** click on **Entire
> Contents** click on **Microsoft Windows Network** click on
> **jgi_psf** click on **Octopus** click on **ProdSeq** click on
> **RCA** click on **Calibration** click on **Cavro Calibrations**
> select **Cavro 3** worksheet.*

6.  If weights are all within the range, continue to dispense all the
    plates in the batch. Acceptable dispense range for 9ul of
    ET-Terminator chemistry is **3.415-3.497g**.

> ***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}**

> 1.1 Remove the DYEnamic ET-Terminator Sequencing Premix Kit and
> respective FW/RV
>
> primers from the -20°C freezer #202 located in Rm 140.
>
> 1.2 Let the reagents thaw on ice.
>
> ***NOTE 1:** Make sure to keep different primers in different ice
> buckets.*
>
> ***NOTE 2:** The ET-Terminator Kit usually takes 30 minutes to thaw.*

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:

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a.  Mark the side of these barcodes with a purple stripe, making sure
    not to cover the barcode itself.

b.  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).*

1.  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.  Match the source plates with their corresponding destination plates
    (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.*

1.  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.  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).*

3.  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.

4.  Open the **Aliquot** **AUTOSTART1** program on the computer monitor.

5.  Run the **WASH_100μlx3** program.

6.  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.*

7.  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 the 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.*

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

9.  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.*

10. Remove source and destination plates from the Hydra-Twister
    stackers.

11. Re-open **Aliquot AUTOSTART1** and run the **WASH_100μlx3** program.

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

12. 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 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: *MB 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 2
> weeks, throw them away in the autoclave waste container.*

1.  For the FW and RV plates:

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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 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. 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.

    1.  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.

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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**.

    1.  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 by pushing down on the red emergency stop button
    located next to the Hydra Twister.

c.  Log off of the computer.

######### Sequencing Chemistry

1.  Wrap two 250-ml bottles in foil and label *FW* or *RV*, and place
    them on ice.

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

> **NOTE 1:** When dispensing on Cavro 1 -- make enough cocktail for 3
> extra plates of FW and RV so the syringes don't run dry. When
> dispensing by hand -- make 20% extra cocktail for each.
>
> **NOTE 2:** Always make fresh ET-Terminator cocktail, never use
> recycled cocktail.

3.  Cap each FW and RV ET-Terminator cocktail bottle and mix by swirling
    gently and inverting each bottle 3 to 4 times.

4.  Place two 250-ml double pharmaceutical graduates wrapped in foil
    into two 1000-ml beakers of ice labeled FW and RV.

5.  Remove foil from the opening of the double pharmaceutical graduates.
    Pour the FW cocktail into the 250-ml double pharmaceutical graduate
    located in the FW beaker and pour the RV cocktail into the 250-ml
    pharmaceutical graduate located in the RV beaker.

6.  Using Cavro 1, dispense **9μl** of the FW and RV ET-Terminator
    cocktail into each well of corresponding FW and RV 384-well plates:

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a.  Log onto the computer for Cavro 1.

b.  Click on the **Cavro 1 Barcodes** icon located on the desktop.

c.  Check the Cavro 1 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 double pharmaceutical graduate.

g.  Make sure the FW stopper is at the bottom of the double
    pharmaceutical graduate.

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 ET-Terminator
> cocktail and contain no air bubbles*

k.  Dump the FW and RV trough contents back into corresponding FW and RV
    double pharmaceutical graduates of ET-Terminator 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
> **9**μl 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 get the problem resolved.*
>
> ***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.*

7.  Purge the leftover ET-Terminator cocktail from Cavro 1 after each
    batch:

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

b.  Pour the leftover ET-Terminator cocktail remaining in the troughs
    down the sink.

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

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

10. 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.*

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.

    1.  Place the plates on the correctly matched thermocyclers.

    2.  Run the **ET-Terminator** 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.*

3.  Wash Cavro 1 after each batch.

> a\. Place the double pharmaceutical graduates in the autoclave bin.
>
> b\. Place the 500-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 500-ml bottle when the water levels
    get low.

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

    1.  After the thermocycler program ends (approx. 2 hours later),
        remove the paired plates from the thermocyclers and place them
        in a bin labeled: *MB batch \# and date.*

> ***NOTE:** Stop the thermocyclers either manually or by computer. 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.***

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

3.  At the end of the day:

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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 500-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 1 Barcodes** program.

g.  Log off of the computer.

# Reagent/Stock Preparation

**[DYEnamic ET-Terminator cocktail, 10μl reaction for Plasmid
Template]{.underline}**

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

1.92 ml Milli-Q Water

1.536 ml DYEnamic ET-Terminator mix

14. μl Primer (250μM)

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1.  When preparing a fresh cocktail mix, prepare enough cocktail for the
    number of source plates in your sample plus 3 additional plates
    according to the *cocktail volume per number of plates* chart
    located in the Sequencing Chemistry bay and also at the end of this
    protocol in **Appendix B**.

2.  Wrap two 250 ml bottles with Aluminum foil and place bottles in an
    ice bucket. Mark each bottle with FW or RV for the two different
    primer directions.

> ***NOTE:** The ET-Terminator is light sensitive*.

3.  Add water, ET-Terminator, and primer to each 250-ml bottle and mix
    by swirling and inverting the bottle.

> ***NOTE:** Keep all reagents on ice while preparing cocktail and
> dispensing into the 384-well plates.*

**[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 ETMB30**

> **95^o^C for 30 seconds**
>
> **95^o^C for 25 seconds**
>
> **50^o^C for 10 seconds 30 cycles**
>
> **60^o^C for 2 minutes**
>
> **Hold at 4^o^C**

Volume = 10μl

**[Cavro 1]{.underline}**

**Setup:**

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

2.  Double-click on the **Cavro 1 Barcodes** **e55A** icon.

**Calibration:**

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

**Shut-down:**

1.  Close the **Cavro 1 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.

# 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

  ----------- -------- -------- ---------- ---------- ----------- -------- --------- ----------
                       ** **               **ET                                       
                                           cocktail                                  
                                           Recipe                                    
                                           (MB)**                                    

  **Plate**   **H2O    **KIT    **Primer   ** **      **Plate**   **H2O    **ET Kit  **Primer
              (mL)**   (mL)**   (uL)**                            (ML)**   (mL)**    (UL)**

  1           1.92     1.536    6.144                 37          71.04    56.832    227.328

  2           3.84     3.072    12.288                38          72.96    58.368    233.472

  3           5.76     4.608    18.432                39          74.88    59.904    239.616

  4           7.68     6.144    24.576                40          76.8     61.44     245.76

  5           9.6      7.68     30.72                 41          78.72    62.976    251.904

  6           11.52    9.216    36.864                42          80.64    64.512    258.048

  7           13.44    10.752   43.008                43          82.56    66.048    264.192

  8           15.36    12.288   49.152                44          84.48    67.584    270.336

  9           17.28    13.824   55.296                45          86.4     69.12     276.48

  10          19.2     15.36    61.44                 46          88.32    70.656    282.624

  11          21.12    16.896   67.584                47          90.24    72.192    288.768

  12          23.04    18.432   73.728                48          92.16    73.728    294.912

  13          24.96    19.968   79.872                49          94.08    75.264    301.056

  14          26.88    21.504   86.016                50          96       76.8      307.2

  15          28.8     23.04    92.16                 51          97.92    78.336    313.344

  16          30.72    24.576   98.304                52          99.84    79.872    319.488

  17          32.64    26.112   104.448               53          101.76   81.408    325.632

  18          34.56    27.648   110.592               54          103.68   82.944    331.776

  19          36.48    29.184   116.736               55          105.6    84.48     337.92

  20          38.4     30.72    122.88                56          107.52   86.016    344.064

  21          40.32    32.256   129.024               57          109.44   87.552    350.208

  22          42.24    33.792   135.168               58          111.36   89.088    356.352

  23          44.16    35.328   141.312               59          113.28   90.624    362.496

  24          46.08    36.864   147.456               60          115.2    92.16     368.64

  25          48       38.4     153.6                 61          117.12   93.696    374.784

  26          49.92    39.936   159.744               62          119.04   95.232    380.928

  27          51.84    41.472   165.888               63          120.96   96.768    387.072

  28          53.76    43.008   172.032               64          122.88   98.304    393.216

  29          55.68    44.544   178.176               65          124.8    99.84     399.36

  30          57.6     46.08    184.32                66          126.72   101.376   405.504

  31          59.52    47.616   190.464               67          128.64   102.912   411.648

  32          61.44    49.152   196.608               68          130.56   104.448   417.792

  33          63.36    50.688   202.752               69          132.48   105.984   423.936

  34          65.28    52.224   208.896               70          134.4    107.52    430.08

  35          67.2     53.76    215.04                71          136.32   109.056   436.224

  36          69.12    55.296   221.184               72          138.24   110.592   442.368
  ----------- -------- -------- ---------- ---------- ----------- -------- --------- ----------

# Appendix B

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

**6/07/04** -- Steve Wilson and Jamie Jett performed an audit between
2:30PM -- 6:00PM. 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.

**9/02/04** - Cavro 1 calibration range changed from 3.26g-3.65g to
3.30g-3.50g.

**10/20/04** -- HTS weight ranges have changed. If weights fall outside
0.3366g -- 0.3743g it gets a Warning level, if it falls outside 0.3272g
-- 0.3837g it gets a do not use level.

**10/28/04** -- Taking Cavro calibrations on the first FW & RV plate
using cocktail instead of water. Acceptable weight range is now
3.415-3.497.