### **Fosmid DNA Isolation Protocol**:

### **Agencourt SprintPrep Kit**

> Version Number: 3.0
>
> Production Start Date: 10/08/04
>
> Version 3.0 Date: 10/06/04
>
> Authors: Jamie Jett, Joe Alessi, Dou-Shuan Yang
>
> Reviewed/Revised by: Danielle Mihalkanin, Lena Philip, Mariana Anaya,
> Steve Wilson, Simon Roberts

# Summary

Use of the Agencourt SprintPrep Kit and solid phase reversible
immobilization (SPRI) technology to isolate Fosmid DNA from *E. coli*
cells in preparation for sequencing chemistry.

# Materials & Reagents

+------------------------------+-------------------+-------------------+
| **[Materials/Reage           | **[Vend           | **[Stock          |
| nts/Equipment]{.underline}** | or]{.underline}** | Numb              |
|                              |                   | er]{.underline}** |
| *[Disposables]{.underline}*  | Axygen            |                   |
|                              |                   | 321-22-052        |
| 384-well PCR Plate, White    | Edge Biosystems   |                   |
|                              |                   | 48461             |
| Clear Adhesive Plate Sealers | Kimberly-Clark    |                   |
|                              |                   | 34120             |
| Kimwipes EX-L                | VWR               |                   |
|                              |                   | 2944-080          |
| 384 well Flat Bottom Black   | Agencourt         |                   |
| Costar                       |                   | 001048            |
|                              | Sigma-Aldrich     |                   |
| Parafilm                     |                   | I9030             |
|                              | Sigma             |                   |
| *[Reagents]{.underline}*     |                   | T-8787            |
|                              | Monahan Paper     |                   |
| SprintPrep (1L)              | Company           | 775011            |
|                              |                   |                   |
| 1)  Sprint Prep Solution     | Millipore Milli-Q | \-                |
|     (store bottles at room   | System            |                   |
|     temperature)             |                   | \-                |
|                              | Robbins           |                   |
| 2)  Sprint Prep Activator    | Scientific        | \-                |
|     (store bottles at 4° C)  |                   |                   |
|                              | Robbins           | \-                |
| 100% Isopropanol             | Scientific        |                   |
|                              |                   | \-                |
| Triton X - 100               | Lab-Line          |                   |
|                              | Instruments       | \-                |
| Pure Bright Germicidal       |                   |                   |
| Bleach                       | LBL               | \-                |
|                              |                   |                   |
| Milli-Q water                | \-                | \-                |
|                              |                   |                   |
| *[Equipment]{.underline}*    | Beckman Coulter   |                   |
|                              |                   |                   |
| Plate Positioner             | Titertek (Thermo  |                   |
|                              | Lab Systems)      |                   |
| Hydra                        |                   |                   |
|                              | Fisher Scientific |                   |
| Titer Plate Shaker           |                   |                   |
|                              |                   |                   |
| Metallic Magnetic Magnets    |                   |                   |
|                              |                   |                   |
| Strong Field Magnets         |                   |                   |
|                              |                   |                   |
| Biomek FX                    |                   |                   |
|                              |                   |                   |
| 96-well Multidrop            |                   |                   |
|                              |                   |                   |
| Isotemp Water Bath (202/205) |                   |                   |
+------------------------------+-------------------+-------------------+

# Calibration Check

**[96-well Multidrop]{.underline}**

NOTE 1: This should be done at the beginning of every day during the
setup of the Multidrop.

NOTE 2: Multidrop Cassettes should be cleaned after each shift. Refer to
the **Multidrop Cleanup** instructions listed under **Instrument
Maintenance**.

1.  Tare an empty, test 96-well Costar plate on the balance in Rm139.
    Before you weigh the plate, make sure the balance is leveled; the
    rear bubble is centered.

2.  Install the Sprint RE1 Multidrop cassette into the Multidrop. Refer
    to **Multidrop Setup** listed under **Instrument Maintenance** for
    installation instructions.

3.  Prime the Multidrop by placing the Multidrop tubing in the Milli-Q
    water bottle and pressing "PRIME/PUMP" button until the trough
    becomes three quarters of the way full. Check to see if liquid is
    dispensing evenly from all tips and make sure no tips are clogged or
    partially clogged.

4.  Use the Multidrop to dispense the appropriate amount of water into
    each well of the 96-well Costar plate (RE1: 30μl/well) and reweigh
    the plate. Make sure that water was dispensed into every well.

> ***NOTE:** The setting on the front of the Multidrop should read 30ul
> for the dispensing of the 12 columns.*

5.  Record the value in the Fosmid Microfill-MD calibration log as well
    as the date, operator, instrument, cassette #, plate \# and any
    comments.

    a.  The acceptable calibration range for the fosmid Multidrop is
        **2.904g-3.045g.**

    b.  To open excel spreadsheet click on **Shortcut to Fosmid
        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 **Library** click on **Fosmids** click
        on **Fosmid Microfill-MD Calibration** click on **Fosmid
        MicroFill-MD Daily Plate Weights** select **Fosmid Multidrop**
        worksheet.

    c.  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.

    d.  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). Submit a work request to have the instrumentation
        group fix the faulty cassette.

6.  After the calibration check has been completed, remove the tubing
    from the water bottle and press the "PRIME/PUMP" button to dispense
    the remaining liquid from the tubing.

7.  Leave the cassette attached to the Multidrop for the remainder of
    the shift.

**[Tip QC for Biomek FX]{.underline}**

Perform the tip QC once daily at the beginning of the shift according to
the **Biomek FX Liquid Handler Fosmid QC** SOP. (Perform only at the
beginning of shift 2; the first shift of fosmid processing)

# Procedure

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

1.  **[DNA Isolation]{.underline}**

    1.  Remove one batch of 96-well costar plates (1 batch = 16 plates)
        from the -20°C freezer (Freezer 200 in lab 139) and allow the
        plates 1 hour and 20 minutes to thaw at room temperature before
        processing.

> ***NOTE 1:** Lay single plates out on bench top to thaw. Do not stack
> them.*
>
> ***NOTE 2:** For each additional batch, pull plates from the freezer 1
> hour and 20 minutes prior to loading on Biomek FX for cleanup.*

2.  During the thaw period, scan the plates into the RCA database:

```{=html}
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a.  In the Venonat database, click on **RCA DB**  **Sprint Prep Cleanup
    and Consolidation**  enter in:

    i.  Make sure your initials are selected as the operator.

    ii. Enter in the lot information for the Sprint Solution, the Sprint
        beads, RE1 buffer, and Triton-X lot, Ethanol lot and Isopropanol
        lot.

    iii. Enter in the processing instrument and consolidation machine
         that will be used.

    iv. Scan in A1 quadrant of all 16 Costar plates that will be
        processed in 1 batch. Submit subsequent batches to the database
        as necessary throughout the duration of the shift.

    v.  Click on **SUBMIT**.

    vi. Print the barcodes (for the RCA destination and FW/RV
        destination plates) on the Libraries barcode printer.

b.  Place all of the barcodes on their corresponding 384-well PCR plates
    (opposite side of the letters, including the FW/RV destination
    plates). Seal the top plate of stack of empty plates.

c.  Using a sharpie, write *your initials* and the *fosmid processing
    date* on the long side of the 384-well RCA plate (same side as the
    well numbers).

    1.  Prepare the Biomek for use according to the **Biomek Setup**
        instructions listed under **Instrument Maintenance**.

> ***NOTE:*** *Change the Biomek FX tips every 2-3 days or when they
> become dirty.*

2.  Open the BioMek FX file **SprintPrep_Manual-Imitation_BG_082604a**

3.  Click **Play.**

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6.  Load the thawed Costar plates onto the Biomek FX deck as described
    in the program splash screen.

> ***NOTE 1:** See Appendix Figure 2 for a visual representation of the
> correct loading order.*
>
> ***NOTE 2:** This program will add 69uL of SprintPrep bead solution
> and 73uL of IPA to the cell culture then mix 36 times. It will then
> perform a total of six 70% ethanol washes, and a final aspiration.
> Total time for each Biomek FX per set of 4 96-well plates is \~ 40
> min.*
>
> ***Workflow Note:** While FX's are running, prep Multidrop and Hydras
> for later use.*

7.  When the first plate is finished, the program will stop to allow you
    to remove the Costar plates for drying and prompt to begin
    processing the next set of Costar plates

8.  Using the spare magnetic plate as a backing, pat the washed plates
    against a paper towel until no ethanol residue is left.

9.  Place in the 37°C blowing incubator (top shelf only at the
    rear/side) for 9 minutes to dry.

> ***NOTE 1:*** *Dry the plates 1-2 minutes longer if cracks not visible
> in all wells and record in the fosmid log excel sheet.*
>
> ***NOTE*** ***2***: *Be very careful not to drop plates while removing
> from incubator!*

10. Install the RE1 Multidrop cassette according to the **Multidrop
    Setup** instructions listed under **Instrument Maintenance** and
    perform a calibration check according to the procedure in the
    **Calibration Check** section above.

11. Using the Multidrop with RE1 cassette, dispense **30μl** of RE1 with
    0.0625% Triton-X into each plate.

12. Shake plates on Titer plate shaker at **speed 7 for 7 minutes**.

> ***Checkpoint:** Following the 7-minute shake, the cell pellet should
> be **completely resuspended** so that the mixture appears homogenous
> and has no clumps.*

13. Leave plates on resting shaker and incubate for 5 minutes.

14. Upon completion of the last RE1 buffer dispense for the shift,
    follow the **Multidrop cassette cleaning and removal** instructions
    listed under **Instrument Maintenance**.

**2. [96-well Costar to 384-well Axygen transfer]{.underline}**

1.  Place the 96-well Costar plates (four per 384-well glycerol stock)
    onto metallic magnetic plates and incubate 5 minutes prior to
    aspiration on the Hydra.

2.  Use the Hydra 96 and a plate positioner to transfer **25μl** of each
    quadrant to a clean, labeled 384-well Axygen plate.

    a.  Use Hydra **File 6**.

    b.  Wash the Hydra twice before use with Milli-Q water.

    c.  Change the wash water after each batch of four 384-well plates.

    d.  When filling from the Costar, be sure to use the metallic
        magnetic plates.

    e.  Go to the computer Solstice (at end of Fosmid bay near fume
        hood) and open the program called
        **barcodeCheck_FosmidConsolidation.exe.**

    f.  Each quadrant should be placed on a specific hydra: A1= Hydra
        16, A2= Hydra 23, B2= Hydra 12, B1= Hydra 21.

    g.  Scan the barcode on hydra 21, 22, 23 and then 16.

    h.  Scan the destination plate barcode.

    i.  Press **Enter** on the computer screen.

        i.  If the program indicates that the plates are not in the
            right order or that one of the plates does not belong in the
            set, correct the problem and scan the plates again.

        ii. If the program indicates that the plates are okay to
            process, transfer the plates using the Hydras.

3.  Check if any wells were not transferred. Transfer them by hand if
    necessary from the costar plate.

4.  If bubbles are present in any wells, quickly spin up to **2000 RPM**
    in a centrifuge.

5.  Check that the axygen plates are labeled on the long side of each
    plate with *operator initials* and *date.*

6.  Seal each 384-well plate with a clear plate seal and put them in the
    production sequencing deli in the "Fosmids ready for Chemistry" bin
    in room 140.

# Reagent/Stock Preparation

**[Sprint Prep Solution + Sprint Prep Activator]{.underline}**

NOTE: The Sprint Prep Solution should be stored at room temperature. The
Sprint Prep Activator should be stored at 4° C.

1.  Put the Sprint Prep Activator on theVari-mix rocker to allow the
    beads to go into the solution. Make sure there are no visible clumps
    of beads before use. (Approximately 1-2 days.)

2.  Add Sprint Prep Activator to the Sprint Prep Solution.

3.  Mix gently by inverting the bottle several times.

4.  Label with the *date,* *your initials,* *and the* *time*.

5.  Place in the 37°C incubator for at least 1 hour before use.

6.  Store combined solution at 37°C.

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

1.  Add **1474ml** of 95% ethanol to a 2L-graduated cylinder.

2.  Bring total volume up to **2000 ml** with Milli-Q water.

3.  Transfer to a 1L glass bottle for storage.

4.  Shake well.

**[RE1 with .0625% Triton-X]{.underline}**

![](media/image1.png){width="0.375in"
height="0.3020833333333333in"}***CAUTION!** Wear gloves, safety glasses
and lab coat when preparing RE1 with Triton X-100 solution.* *Make sure
you are making this solution in the fume hood located in lab 139. Keep
Triton X-100 bottle tightly* *closed* *when not in use. Place the used
tips in the Triton X-100 waste container for proper disposal.*

1.  Add **500ml** RE1 to a 500 mL bottle

2.  Add **312.5μl** Triton-X to the RE1

> a\. Make sure to pipette the Triton-X very slowly, as the detergent is
> very viscous

3.  Place in a 37 °C incubator to allow the Triton-X to go into solution
    before use

4.  After removing from the 37°C incubator, invert several times to mix.

5.  Store at room temperature.

# Instrument Setup and Maintenance

**[Biomek FX]{.underline}**

**Setup:**

1.  Turn on 37˚C water bath and add water if necessary. The water bath
    needs to be filled half way.

2.  Turn on the peristaltic pump and set to reverse flow (arrow down) to
    empty tubing of all remaining wash water.

    a.  For the SPRIntPrep beads reservoir, uncap and remove the suction
        tube from the reservoir before turning pump on.

3.  Connecting the reagents

    a.  When the BioMek deck reservoirs and tubing are empty replace the
        Milli-Q wash bottles with appropriate reagent bottles. For 2
        runs (a total of four 384-well plates)

    ```{=html}
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    ```
    i.  Minimum IPA volume in bottle should be **250**ml.

    ii. Minimum 70% EtOH volume in bottle should be **2L**.

    iii. Minimum SPRIntPrep bead solution volume in bottle should be
         **250**ml.

    iv. After connecting, place the SPRIntPrep bead solution bottle in
        the 37-degree water bath.

        a.  Set the peristaltic pump to forward flow and allow the
            BioMek deck reservoirs to fill. Make sure that the pump
            speed is set to 55.

        b.  Allow the reagents to cycle for at least 10 minutes prior to
            use.

4.  Priming the wash station

    a.  Check that the red valve on the left is open.

    b.  Set the wash station pump switches (on the controller device as
        well as the solenoid) to manual on and allow to run for 10
        seconds, then set back to automatic.

**Cleanup:**

If there are no more runs for the day, then the following steps should
be carried out:

1.  Switch the Ismatec pump to the "reverse" direction and prop the
    reservoirs at an angle so that their reagents are fully emptied.
    Stop the pump once lines are empty.

> ***NOTE 1:** For the SPRIntPrep beads reservoir, uncap and remove the
> suction tube from reservoir.*
>
> ***NOTE 2:** If residue remains in the SPRIntPrep deck reservoir, pour
> a small amount of any remaining SPRIntPrep solution into the deck
> reservoir and agitate to put the residue in solution.*

2.  Remove the orange caps from the reagent bottles and replace them
    with the original bottle caps.

3.  Flushing the tubing:

    a.  For each BioMek FX, fill one 500ml bottle and two 1-Liter
        bottles with Milli-Q H2O.

> ***NOTE 1:** Change Milli-Q water rinse bottles daily.*
>
> ***NOTE 2:** May need to fill additional 1L bottles to for additional
> flushing of Sprint tubing.*

b.  Replace each reagent reservoir with an appropriately sized bottle of
    Milli-Q H2O

    i.  For the IPA bottle, replace it with a 500mL bottle of Milli-Q
        water

    ii. For the sprint reagent bottle and the 70% EtOH bottle replace
        them with a 1L bottle of Milli-Q water.

c.  The 1-Liter bottle is intended to replace the 70% EtOH reservoir

d.  Switch Ismatec pump to the "forward" flow direction (arrow up) and
    allow the BioMek deck reservoirs to fill. Continue pumping water
    through for 5 minutes.

e.  Switch set the Ismatec pump to the "reverse" flow direction (arrow
    down), prop the reservoirs at an angle, and allow the BioMek deck
    reservoirs to empty completely.

    i.  For the SPRIntPrep beads reservoir, uncap and remove suction
        tube from reservoir while the pump is in the "off" position and
        before beginning it in the "reverse" direction.

```{=html}
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```
4.  Turn off peristaltic pump and release EtOH and IPA tubing from the
    pump.

5.  Repeat **steps 3.a-e** for Sprint tubing until cycling waste water
    is completely clear.

6.  Turn off the 37°C water bath.

7.  Remove the orange cap from the waste bottle and dispose of the waste
    into the designated red Hazardous Waste Container.

> ***NOTE:** If the waste container is empty, you will need to mark it
> with the date that liquid started being collected in the container.*

8.  Place the waste bottle back on the Biomek.

9.  Close out the software window by selecting **File**  **Exit**. If
    prompted to save changes, click on **NO**.

**Moving the Ismatec Tubing:**

***NOTE:** When moving or changing the Ismatec tubing, make sure the
Biomek is not running.*

1.  Switch the Ismatec pump off.

2.  One at a time, release an Ismatec pump white applicator, dissociate
    it from its tubing, and move the white applicator over one notch.

3.  After all of the white Return and Supply applicators have been
    moved, spray the bottom of the tubing that is now under the white
    applicators with Tri Flow industrial lubricant Switch the Ismatec
    pumps on to the forward direction and spray the rotating device with
    Tri Flow industrial lubricant.

4.  Switch the Ismatec pumps off.

5.  Re-attach the Ismatec pump white applicators and snap down onto the
    Ismatec pump rotating device.

> ***NOTE:** Make sure to place the applicators back in the same order.*

6.  Switch the Ismatec pump on to the forward direction.

7.  Mark that the tubing has been moved.

**Changing the Ismatec Tubing:**

1.  Switch the reagent Ismatec pump to the reverse direction and prop
    the reservoirs at an angle so that their reagents are fully emptied.

2.  Once all reservoirs and tubing are emptied, switch the Ismatec pump
    off.

3.  One at a time, release an Ismatec pump white applicator, dissociate
    it from its tubing, remove the old tubing and place it in the
    autoclave waste.

4.  Replace with new tubing of the same color and re-attach the white
    applicator to the tubing.

> ***NOTE:** All supply lines should have purple and gray tubing; all
> return lines should have purple and white tubing.*

5.  After all of the tubing has been changed, spray the bottom of the
    tubing that is now under the white applicators with Tri Flow
    industrial lubricant.

6.  Switch the Ismatec pump on to the forward direction and spray the
    rotating device with Tri Flow industrial lubricant.

7.  Switch the Ismatec pump off.

8.  Re-attach the Ismatec pump white applicators and snap down the
    applicators onto the Ismatec pump rotating device.

> ***NOTE:** Make sure to place the applicators back in the same order.*

9.  Switch the Ismatec pump on to the forward direction.

10. Place a piece of tape onto the Ismatec pump motor labeled with what
    date the tubing needs to be moved

    a.  For EtOH and Bead tubing: move one week later and change 2 weeks
        later.

    b.  For IPA tubing: move after 3 days and change after 1 week.

**Changing the Masterflex tubing:**

***NOTE:** When moving or changing the MasterFlex tubing, make sure the
Biomek is not running.*

1.  At the beginning of every week, release the MasterFlex tubing
    clamps, move the MasterFlex tubing over one notch, and reclamp the
    MasterFlex tubing clamps. Mark the date it was moved on the tubing.

2.  There are four spaces that the MasterFlex tubing can be moved. When
    all spaces have been filled (approx. 1 month), enter a work request
    to have the MasterFlex tubing changed by the Instrumentation group.

**Daily Maintenance**:

***NOTE:*** *All reagent and water bottles should be labeled with
operator initials and date.*

[Sprint Prep Solution]{.underline} [and RE1]{.underline}: New glass
reagent bottles (500ml)should be filled from plastic stock bottles when
the current one runs out or if the solution appears abnormal.

[IPA]{.underline}: New glass reagent bottles can be refilled from the
glass stock bottles for one week. Completely new IPA reagent bottles
with fresh IPA should be made at the beginning of each week.

[70% EtOH]{.underline}: Fresh 70% ethanol should be made daily in new
glass reagent bottles.

[Milli-Q water]{.underline}: For cleanup, fresh water bottles should be
prepared daily.

**[Multidrop]{.underline}**

**Setup:**

**Installation of Sprint RE1 cassette:**

1.  Perform a visual check on the Sprint RE1 multidrop cassette and the
    cassette tubing to make sure that they are undamaged.

2.  Take the lower part of the cassette into your right hand with the
    dispensing cassette tips pointing down and the upper part in your
    left hand.

3.  Carefully place 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.

4.  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.

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

6.  Make sure that the tension limiting wires have a loose fit around
    the rotor shaft and the tubes are evenly placed on the rotor needles
    (four tubes on each half of the pump rotor). Inspect to ensure that
    the tubing is not kinked or overlapping.

7.  Pull rotor cover over the rotor.

**Priming with water then RE1 plus Triton-X:**

1.  Prime the Multidrop by placing the Multidrop tubing in the warm
    Milli-Q water bottle and pressing **PRIME/PUMP** button until the
    trough becomes three quarters of the way full. Check to see if the
    liquid is dispensing evenly from all tips and make sure no tips are
    clogged or partially clogged.

***NOTE:** If the tips are clogged, see **Clogged Tips** instructions
below.*

2.  Remove the Multidrop tubing from the Milli-Q water bottle and
    continue pressing the **PRIME/PUMP** until no more liquid is flowing
    out of the tubing. Some residual droplets may remain inside the
    tubing.

3.  Prime the multidrop by placing the multidrop tubing in the
    RE1-Triton-X bottle and pressing **PRIME/PUMP** button until all
    tips are dispensing buffer evenly.

4.  Check to see if the liquid is dispensing evenly from all tips and
    make sure no tips are clogged or partially clogged.

**Clogged Tips:**

> If the tips are clogged, insert a small metal wire into the tip to
> remove the clog. As needed, try priming the tubing with hot water
> followed 70% ethanol, but rinse again with Milli-Q water before using
> the buffer reagents. Run a test plate through to check for even
> dispensing. If the tips are still clogged, change to the backup
> multidrop cassette and submit an instrumentation work request.

**Cleanup (RE1 cassette):**

> After dispensing of RE1 buffer is completed for the day, prime the
> cassette tubing with warm Milli-Q water. Gently wipe the multidrop
> cassette with 70% ethanol using Kimwipes prior to putting each
> cassette back in its box.

**RE1 Cassette Removal and cleanup:**

1.  Upon completion of buffer dispensing, remove the multidrop tubing
    from the solution and press the **PRIME/PUMP** button until all of
    the solution has been dispensed from the tubing.

2.  Clean the multidrop tubing by priming it with Milli-Q water until
    the trough becomes three quarters of the way full. Remove the tubing
    from the water and continue dispensing until no water is left inside
    the tubing.

3.  Push the rotor cover off of the rotor.

4.  Gently remove the upper part of the multidrop cassette, followed by
    the lower part.

5.  Gently wipe the multidrop cassette with 70% ethanol using the
    Kimwipes.

6.  Place the cassette in its designated case.

**Weekly Maintenance**

Calibration check of each cassette. Refer to Calibration Check.

**Hydra Maintenance**

1.  Once per week, place a trough with Coulter Clenz cleaning reagent in
    the destination area and run through the following steps. Use **File
    2** on the Hydra.

    a.  Cycle through the Menu to Asp/Disp.

    b.  Hit the Fill button to Fill the needles to 50uL then let them
        soak for 1-2mins.

    c.  Empty the needles.

    d.  Cycle through the Menu to Wash and run the program. This will
        wash the needles 5 times.

    e.  Remove the Coulter Clenz trough.

    f.  Place a trough with MilliQ Water in the destination area.

    g.  Cycle through the Menu to Wash and run the program. This will
        wash the needles 5 times.

    h.  Replace Water trough after each set of 5 washes.

    i.  Repeat this method 3 times (total of 15 washes). Steps "*f*" to
        "*i*".

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

    a.  Fill the needles **50**% full with MilliQ water.

    b.  Turn off the instrument by using the switch located on the back
        of the Hydra next to the power cord.

# 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

**Figure 1. FX Deck Layout**

> ![](media/image2.png){width="6.748425196850394in"
> height="5.252755905511811in"}