### **Fosmid DNA Inoculation Protocol**:

### **Agencourt SprintPrep Kit**

> Version Number: 3.2
>
> Production Start Date: 10/08/04
>
> Version 3.2 Date: 12/14/04
>
> Author(s): Jamie Jett, Joe Alessi, Dou-Shuan Yang
>
> Reviewed/Revised by: Danielle Mihalkanin, Lena Philip, Mariana Anaya,
> Simon Roberts, Steven Wilson

# Summary

Samples from fosmid glycerol stocks are inoculated into 96-well costar
plates containing 2-YT broth in preparation for fosmid isolation using
the Agencourt Sprint Fosmid Prep Kit.

# Materials & Reagents

+------------------------------+-------------------+-------------------+
| **[Materials/Reage           | **[Vend           | **[Stock          |
| nts/Equipment]{.underline}** | or]{.underline}** | Numb              |
|                              |                   | er]{.underline}** |
| *[Disposables]{.underline}*  | VWR               |                   |
|                              |                   | 27442-396         |
| 96-well 300ul Costar Plate   | Qiagen            |                   |
|                              |                   | 19571             |
| AirPore Tape Sheets          | Kimberly-Clark    |                   |
|                              |                   | 34120             |
| Kimwipes EX-L                | VWR               |                   |
|                              |                   | 2944-080          |
| 384-well Flat Bottom Black   | Teknova           |                   |
| Costar                       |                   | Y0167             |
|                              | Monahan Paper     |                   |
| Parafilm                     | Company           | 775011            |
|                              |                   |                   |
| *[Reagents]{.underline}*     | Millipore Milli-Q | \-                |
|                              | System            |                   |
| 2X-YT broth                  |                   | 857440-25G        |
|                              | Aldrich           |                   |
| Pure Bright Germicidal       |                   | A3256             |
| Bleach                       | Sigma-Aldrich     |                   |
|                              |                   | \-                |
| Milli-Q water                | S                 |                   |
|                              | afeway/Albertsons | \-                |
| Chloramphenicol              |                   |                   |
|                              | Robbins           | \-                |
| L-arabinose                  | Scientific        |                   |
|                              |                   | \-                |
| McCormick Blue Food Color    | Lab-Line          |                   |
| Dye                          | Instruments       | \-                |
|                              |                   |                   |
| *[Equipment]{.underline}*    | Matrix            |                   |
|                              |                   |                   |
| Plate Positioner             | Fisher Scientific |                   |
|                              |                   |                   |
| Titer Plate Shaker           |                   |                   |
|                              |                   |                   |
| PlateMate                    |                   |                   |
|                              |                   |                   |
| Isotemp Water Bath (202/205) |                   |                   |
+------------------------------+-------------------+-------------------+

# Calibration Check

**[Tip QC for Platemate 2]{.underline}**

1.  A tip QC should be performed using blue dye and the Spectramax 384
    Plus once per day at the beginning of the shift. Refer to the **QC
    Protocol for the PlateMate Liquid Handler:Fosmid Inoculations**. The
    entire set of tips should be changed before running the Tip QC, and
    if there is any clogging or error with specific tips. Repeat Tip QC
    if additional tips are changed.

**[Microfill Calibration]{.underline}**

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

NOTE 2: Make sure the Microfill is on the correct dispensing program,

NOTE 3: Refer to **Operating the Fastrack Crane & Microfill** at the end
of the protocol for proper Priming, Dispensing, and Purging techniques.

1.  Tare an empty test 96-well costar plate on the balance in Rm139.
    Before the plate is weighed, make sure the balance is even and
    centered.

2.  After the Milli-Q water-priming step is completed, follow the steps
    listed in the **Priming and Dispensing 2-YT (with chloramphenicol
    and L-arabinose) broth** section under **Operating the Fastrack
    Crane & Microfill** to dispense Milli-Q water into the 96 wells of
    the tared test Costar plate.

> ***NOTE:** Check plate to make sure that water was dispensed evenly
> into every well. If dispensing appears uneven across the wells, repeat
> **steps 1-2.** If the dispensing continues to be uneven, stop using
> the instrument and submit a work request immediately.*

3.  Reweigh the plates on the balance.

> ***NOTE:** The acceptable calibration range for microfill 2 is
> **14.163g-17.718g**. The acceptable calibration range for microfill 3
> is **13.817g-14.578g.***

4.  Record these values on the Fosmid Microfill-MD Calibration log
    sheet.

    a.  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 **RCA** click on **Calibration** click
        on **Fosmid Microfill-MD Calibrations** click on **Fosmid
        MicroFill-MD Daily Plate Weights** select **Microfill 2**,
        **Microfill 3**, or **Multidrop** worksheet.

    b.  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 instrument 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.

    c.  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, stop using
        the instrument and submit a work request immediately.

**[Platemate and Microfill Sterility:]{.underline}**

1.  Platemate: Inoculate one sterility block at the end of the last run.
    Follow **step 1.22** of the procedure. Label plate with *operator
    initials, date, and platemate sterility.*

2.  Microfill: Dispense 2x-YT broth into one extra costar plate for each
    Microfill at the end of dispensing. Label plate with *operator
    initials, date, and microfill 2 or microfill 3 control plate.*

3.  Incubate all three plates in ATR Multitron for 20 hours with
    production sample plates.

4.  Remove all three plates from ATR Multitron following incubation and
    check for growth. Record results. If there is any growth in the
    plates, immediately send an email to supervisor and fill out an
    incident report. Record all affected plates in the work request.

    a.  If the Microfill fails sterility, submit a work request
        immediately to have Instrumentation change the manifold and
        syringe pump. Re-run the sterility test and begin using the
        instrument.

    b.  [If the platemate fails sterility, continue using, as tips are
        changed daily.]{.underline}

# Procedure

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

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

1.  Check the sterility plates for growth. Refer to the **Platemate and
    Microfill Sterility** procedure above.

2.  Remove inoculation glycerol stocks from the -80°C freezer. Thaw the
    first set of 6 glycerol stocks at 37˚C for 1 hour. Thaw the rest of
    the glycerol stocks at room temperature for 2 -- 2.5 hours and then
    store at 4˚C until they are loaded onto the platemate.

3.  During the thaw period, scan the glycerol stocks into the RCA
    database:

```{=html}
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```
a.  In the Venonat database, click on **RCA DB**  **Scan Sprint Plates
    For Glycerol Transfer**  enter in:

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

    ii. Select the PlateMate instrument you will be using.

    iii. Enter in the lot information for the 2-YT broth.

    iv. Enter lot information for Antibiotic and Arabinose.

    v.  Enter in the ATR Multitron shaker incubator that the plates will
        be loaded into.

    vi. Scan in all of the glycerol stock barcodes (up to 25plates at a
        time).

    vii. Click on **SUBMIT**.

    viii. Print the barcodes (to be used on the costar plates) on the
          Libraries barcode printer.

> ***NOTE:** When printing barcodes, make sure that the printer is
> "Online". Once your barcodes have printed press **Online** again to
> take the machine off line and then **Feed** in order to give enough
> barcodes that you can tear the strip off. If you do not take the
> barcode printer offline before depressing Feed, the printer will print
> multiple copies of your last barcode.*

b.  Place the barcodes on each of the costar plates (opposite of the
    lettered side).

    1.  Using a thoroughly cleaned Microfill, dispense 150μl of 2-YT
        broth with 12.5 μg/ml Chloramphenicol and 0.02% L-Arabinose into
        96-well 300ul Costar Plates:

```{=html}
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```
a.  Run the 2% bleach prime program (Refer to **Priming with 2% Bleach**
    section under **Operating the Fastrack Crane & Microfill**).

b.  Run the water prime program (Refer to **Priming with Milli-Q water**
    under **Operating the Fastrack Crane & Microfill**) three times.

c.  Run the "Trough Prime" program (Refer to **Priming and Dispensing
    2-YT (with chloramphenicol and L-arabinose) broth** under
    **Operating the Fastrack Crane & Microfill**).

> ***NOTE:** Each new bottle of broth should get a new autoclaved
> cap-tube fitting.*

d.  Dispense broth into one extra costar plate to be used as a negative
    control.

e.  Do not leave the broth bottles attached to the instruments with
    broth sitting in the tubing for prolonged periods of time. If it is
    necessary to leave the instruments idle while in the middle of
    dispensing, prime them through with 2% bleach and water.

f.  Upon completion of the 2-YT broth dispensing, follow the microfill
    cleanup instructions listed under **Operating the Fastrack Crane &
    Microfill**.

    1.  Turn Platemate 2 on and allow the homing procedure to finish.

    2.  Prime the wash station according the to protocol listed on the
        front of the Platemate.

    3.  Check to make sure that the water supply reservoir is filled
        with Milli-Q water and that the waste reservoir is empty.

    4.  Make up more bleach if necessary.

    5.  Open the ControlMate 1.1.0 program

    6.  Change the pipette tips at the beginning of the shift each day:

        a.  Using the Add-Ins/Change Pipette or Tips Menu, select tips
            only.

        b.  Click **Start**

        c.  Remove the old Tip Magazine.

        d.  Click **Done**

        e.  Insert a new Tip Magazine.

        f.  Click **Done**

    7.  Perform a Platemate QC calibration daily as described in the
        **QC protocol for the PlateMate Liquid Handler: Fosmid
        Inoculations.**

    8.  Add approximately **135ml** of fresh 2% bleach to the bleach
        reservoir.

> ***NOTE:** Make up a fresh 2- liter bottle of 2% bleach daily*.

9.  Check to make sure that the water supply reservoir is filled with
    Milli-Q water and that the waste reservoir is empty.

10. Load the glycerol stock plates into Left Stacker B (Start Source).

> ***NOTE:** 1 batch = 6 glycerol stock source plates*

11. Load the filled 96-well 300ul Costar plates into Right Stacker B
    (Start Destination). Load the Costar plates appropriately to match
    the order of glycerol stock plates.

> ***NOTE 1:** 1 batch = 24 destination costar plates*
>
> ***NOTE 2***: *See Appendix 1 for a visual representation of the
> correct loading order*.

12. Open the Platemate program **Fosmid Inoculation into [300ul Falcon
    SW 121404.cms]{.underline}**

> ***NOTE:** This program will transfer 6μl of glycerol stock from a
> 384-well Nunc plate to four 96-well 300ul Costar plates.*

13. Set the number of glycerol stock plates for inoculation.

    a.  Click on the transfer step

    b.  Set the number of iterations = to number of glycerol stocks.

    c.  Except for the last batch of the day, uncheck the sterility
        block step.

> i\. For the last batch of the day, add the additional sterility block
> costar plate to the stack and check the sterility block step.

14. Click on **Play**

15. Click on **Start** when ready.

> ***Important!** Do not leave the Platemate's vicinity while it is
> running. You must continuously monitor the Platemate in case a failure
> occurs during the run.*

16. When the run is finished, the barcodes must be checked for accurate
    processing. A barcode checking form should appear automatically at
    the end of the run. If an error box appears, contact an
    Instrumentation software developer. Press the **Proceed** button to
    see more barcodes. Select which dispenser was used, operator
    initials and then press **Save.** Press **Archive** to complete the
    transaction.

    a.  If there is a mix-up, the screen will be red. Contact a group
        supervisor or instrumentation engineer before continuing. A
        mix-up will require a comment for the mixed-up barcodes as well
        as a batch comment at the bottom of the window.

    b.  A yellow screen indicates that there was a plate with a bad
        barcode format or non-read. Scan in the appropriate barcode or
        fail the plate. Be sure to fill in a comment for the reason of
        failure.

17. Remove the glycerol stocks from Left Stacker A (End Source) and seal
    with aluminum foil lids using the automated pneumatic sealer. Place
    in the --80°C freezer for re-racking.

18. Setting aside plates for QC Stamping:

    a.  Set aside 4 glycerol stock plates in the 4° C Fosmid Deli in a
        bin labeled "For QC Stamping."

    b.  These plates should include a random sampling of libraries that
        were inoculated.

> ***NOTE:** QC will pick up plates for stamping after plates have been
> inoculated.*

19. Remove the 96-well 300ul Costar Plates from Right Stacker A (End
    Destination) and seal all Costar plates with AirPore tape sheets.

> ***NOTE:** All tape sheets should be in the same direction.*

20. After 6 glycerol stocks, unplug the bleach reservoir and empty into
    container labeled bleach waste.

21. Repeat **step 1.11-1.23** until all glycerol stocks have been
    inoculated for the shift.

> ***NOTE:** For the last batch of the day, make sure to check the
> sterility block option.*

22. Place the Costar plates into the **37°C ATR Multitron incubator at
    85% humidity, shaking at speed 600**.

> ***NOTE:** Turn temperature and humidity controls on 30 minutes prior
> to use, and ensure that the DI water reservoir is filled to the line
> with DI water.*

23. Set the timer located on top if the incubator for 20 hours. Incubate
    in the **ATR** for **20 hours.** Remove plates when the alarm
    sounds.

24. Remove the cell cultures from the ATR and freeze at -20°C for at
    least 16 hours before processing.

# Reagent/Stock Preparation

**[10% L-arabinose]{.underline}**

1.  To a 50mL conical tube add **3g** L-Arabinose and bring up to
    **30ml** using Milli-Q water.

2.  Mix thoroughly by shaking.

3.  Transfer 2 ml aliquots to labeled 2 ml Eppendorf tubes.

4.  Freeze at -20°C.

**[2-YT broth with 12.5μg/ml Chloramphenicol and 0.02%
L-arabinose]{.underline}**

1.  Add **368μl** of 34μg/ml Chloramphenicol to 1L of 2-YT broth.

2.  Add **2ml** of 10% L-Arabinose solution to 1L of 2-YT broth.

3.  Mix gently by inverting the bottle several times.

**[Antibiotic Preparation and storage:]{.underline}**

> Chloramphenicol solution (34mg/ml): Add **0.51g** of chloramphenicol
> to **15ml** of 100% ethanol in a 15-ml Corning tube covered with foil
> tape. Store at -20˚C.

**[2% Bleach]{.underline}**

1.  Add **20ml** of germicidal bleach to a 1L graduated cylinder.

2.  Bring total volume up to **1000ml** with Milli-Q water.

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

4.  Shake well.

**[Blue Dye Stock Solution for Tip QC]{.underline}**

1.  Using a graduated cylinder, measure out **400ml** of Milli-Q water.

2.  Pour the water into a 500ml bottle.

3.  Use a 1-ml pipettor to pipette **2ml** concentrated dye into the
    bottle in 1ml increments.

4.  Rinse the pipette tip 4x in the bottle solution after each 1ml
    addition to ensure complete transfer of viscous concentrated dye.

5.  Label bottle: *QC Blue Dye (Fosmid Inoc),* *date prepared and your
    initials*.

6.  Store at room temperature.

# Operating the Fastrack Crane & Microfill

**[Microfill]{.underline}**

**Switching On:**

1.  Switch the instrument on by using the main power switch on the rear,
    right-hand side of the microfill. The microfill will self-test then
    go to the main menu.

**Instrument Setup:**

1.  Fill two labeled 1L bottles with fresh Milli-Q water (one for each
    microfill) and place each bottle on the same horizontal as its
    corresponding dispenser to ensure optimal pump performance.

2.  Fill one labeled 500ml bottle with 2% Bleach.

3.  Cap all reagent bottles until they are "hand-tight". Do not over
    tighten.

**Priming with 2% Bleach**

1.  Replace the 2% Bleach bottle cap with the orange cap-tube fitting
    (found on the bleach bottle from the previous day's use) and connect
    to the microfill. Make sure that the vent hole is not covered.

2.  Select the **RUN** button from the Main Menu screen.

3.  Select **PRIME**.

4.  Pre the **OPTIONS** button until "120ML" method appears on the
    screen.

5.  Select **ENTER.**

6.  Select **START**. Perform the bleach priming once.

> ***NOTE:** The 120ML method will prime 120ml of bleach.*

7.  Press the **MAIN MENU** button to return to the Main Menu display.

8.  Disconnect bleach bottles cap-tube fitting from the microfill.

**Priming with Milli-Q water**

1.  Replace the Milli-Q water bottle cap with the orange cap-tube
    fitting (found on the Milli-Q bottle from the previous day's use)
    and connect to the microfill. Make sure that the vent hole is not
    covered.

2.  Select the **RUN** button from the Main Menu screen.

3.  Select **PRIME**.

4.  Press the **OPTIONS** button until "200ML" method appears on the
    screen.

5.  Select **ENTER**.

6.  Select **START**. Perform the water priming three times.

> ***NOTE:** The 200ML method will prime 200ml of water.*

7.  Press the **MAIN MENU** button to return to the Main Menu display.

8.  Disconnect the water bottle's cap-tube from the microfill.

**Priming and Dispensing 2-YT (with chloramphenicol and L-arabinose)
broth:**

1.  After the addition of the chloramphenicol antibiotic and L-arabinose
    inducer to the 2-YT broth, replace 2-YT bottle cap with an
    autoclaved, white cap-tube fitting (found in the fosmid lids drawer)
    and connect to the microfill. Make sure that the vent hole is not
    covered.

> ***NOTE:** Make sure that the tubing reaches the bottom of the 2-YT
> bottle.*

2.  Select the **RUN** button from the Main Menu screen.

3.  Select **PRIME**.

4.  Press the **OPTIONS** button until "Trough_Prime" method appears on
    the screen.

> ***NOTE:** The Trough Prime method will prime 17ml of broth* *and
> establishes communication between the microfill and the robot arm.*

5.  Select **ENTER**.

6.  Select **START**.

7.  Press the **Main Menu** button to return to Main Menu display.

**Stacker Setup**

1.  Place the correct number of 96-well Costar plates in the appropriate
    stacker for the method chosen.

2.  Make sure that all of the plates are in the same orientation and
    aligned properly.

3.  Do not add or remove plates from stackers during a run.

  -------------- ---------- ----------- ----------- ----------- -----------
  **Method       **\#       **Stacker   **Stacker   **Stacker   **Stacker
  Name**         Plates**   1**         2**         3**         4**

                                                                

  Costar 96      up to 70   20 or less  20 or less  20 or less  20 or less
  -------------- ---------- ----------- ----------- ----------- -----------

**Running PlateCrane Application**

1.  Double click on the PlateCrane software icon on the desktop located
    next to the microfill.

2.  Click on **NO** when the *Run system SIMULATE MODE?* screen appears.

3.  Click on **Run Methods (F1)**.

4.  Click on **Start Run**.

5.  Select the appropriate "Costar 96" method to run.

6.  Click on **Continue** when the method has been chosen.

7.  Click on the correct source stackers and then click on **ENTER**.

> ***NOTE:** The source stackers are highlighted in white.*

8.  When asked *Is the Method List complete?*, click on **YES**.

9.  Press the Space Bar on the keyboard to initiate the run.

10. ![](media/image1.png){width="0.375in"
    height="0.3020833333333333in"}When asked *Is the PLATE CRANE clear
    to HOME now?,* Click on **YES** only if there are no obstructions
    above the stackers.

> ***CAUTION!** ROBOT CAN MOVE FAST -- KEEP CLEAR*.

11. Run will start once robot has homed to the rear of the table.

12. Once the method has been completed, the robot will home itself and
    remain there.

13. Remove plates by lifting stacker off its base and sliding the plates
    out of the stacker.

14. Place the costar plates in stacks of 24 with a clear plate seal on
    the top plate.

15. Label cell culture plates with the appropriate barcodes for
    inoculation.

**Cleanup**

1.  Cleanup Guidelines:

    a.  Back to back batches is considered 0 time through 1 hour, and no
        bleach wash with water rinse would be required.

    b.  At a time greater than 1 hour, the operator is required to
        complete a bleach wash and water rinse. The procedure for doing
        this is in the setup and below.

    c.  At the end of the shift, a bleach wash and water rinse is
        required before shutting down the instrument.

```{=html}
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```
2.  After the Microfill has been used, decontaminate the Microfill
    cap-tube fitting from the used LB broth bottles by rinsing with 70%
    ethanol using a squirt bottle.

3.  Remove the Microfill cap-tube fitting and place it in the
    dishwashing bin. The caps should be cleaned and autoclaved by the
    operations technician.

4.  Connect the microfill to the bleach bottle cap. Make sure that the
    vent hole is not covered.

> ***NOTE:** Connect the short Y-Tube fitting to the bleach bottle cap.*

5.  Following the **2% Bleach Priming** instructions about to prime 2%
    bleach though the Microfill.

6.  Disconnect the bleach bottle's cap-tube fitting from the Microfill
    and connect it with the water bottle.

7.  Following the **Water Priming** instructions above to prime water
    through the Microfill.

8.  Waste from the Multidrop should be disposed of into SAA Flam Can A,
    while waste from the Microfill Plate Crane can be disposed of down
    the sink. Waste collected from cleaning the cap-tube fittings should
    also be disposed of into SAA Flam Can A.

9.  Check the waste container for level of waste accumulated. If the
    container is 3 quarters full, empty it into the red Hazardous Waste
    containers. The waste container should be emptied daily.

10. Leftover LB broth should be poured down the sink followed by a small
    amount of bleach and the LB bottles should be placed in the
    autoclave waste.

11. Retain orange caps from stock reagent bottles daily. They will be
    changed out during bi-weekly maintenance according to the
    **Microfill Preventative Maintenance** protocol.

12. Leftover 2-YT broth should be poured down the sink and the 2-YT
    bottles should be placed in waste.

13. Clean rails, external surfaces, and the surrounding area with 70%
    EtOH using a spray bottle.

# Troubleshooting

**[Cell Growth/Evaporation]{.underline}**

1.  After the freezing step, each well should have a visible clump of
    cells

2.  If suspicious, test the cell growth using OD 600. Refer to the
    procedure in the Inoculation Protocol.

    a.  Take Costar plates directly from the incubator after the growth
        step.

    b.  Select an assortment of plates.

    c.  Using the Microdrop, fill an appropriate number of 96 300ul
        Costar Plates with 95 ul LB broth, as well as a blank plate with
        100 ul LB broth.

    ```{=html}
    <!-- -->
    ```
    i.  Make sure to use the cassette labeled 2-YT broth.

    ii. Empty and clean according to Microdrop instructions.

    ```{=html}
    <!-- -->
    ```
    d.  If possible, place the selected growth stock plates onto a Titer
        plate shaker at speed 6 to keep the cells resuspended.

    e.  Using BioMek FX7 (the R and D BioMek in Room 458)

```{=html}
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```
i.  Clear all labware except for waste reservoir off the deck.

ii. Prime the wash stations.

iii. Place Beckman Coulter P20 tips underneath each pod.

iv. Open Program **Jamie 5ul transfer P20.bmt**

v.  Click **Play**

vi. Load the plates onto BioMek FX5 according to layout presented.

vii. Click **Play**

> ***NOTE**: This program will transfer 5 ul of growth stock into 95 ul
> of LB broth, making a 1:20 dilution of cells.*

a.  Test 16 well volumes randomly across each original growth plate
    using a P200 pipette

> ***NOTE:** Expect the well volumes to be around 125-30 ul, with a
> fluctuation of about 10 ul both above and below.*

i.  Remember to compensate for the 5 ul removed to take OD 600.

ii. Record these numbers in a spreadsheet and average for each growth
    plate.

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```
f.  Place 1:20 dilution plates on a Titer plate shaker at speed 6 to
    keep the cells resuspended.

g.  Using a Spectramax, go to the Softmax Pro 3.1.2 program (or SPF312
    shortcut).

    i.  Open **Assays** -- **Basic Protocols**

    ii. Select **Raw Optical Density**

    iii. Set **Wavelength (Lm1) to 600**, data mode to **absorbance**,
         and **automix for 15 seconds** once.

    iv. Take O.D. values for each plate and save to an Excel Spreadsheet

1\) Take O.D. values for the blank as well and save to the Excel
spreadsheet.

> 3\. Calculating Cell Count

a.  For each plate, subtract each well's blank value from the
    corresponding well's OD600 result.

b.  Multiply these results by 20.

c.  Multiply these results by the mean well volume across the plate.

d.  Multiply each well's result by 5E+05.

e.  This value is the approximate cell count for the well.

> ***NOTE**: Expect cell count values of 1.5E+08 cells per well, with a
> variance of 5E+07.*

# 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. Platemate 2 Setup.