Plating of Transformation Stocks Protocol

> Version Number: 4.2
>
> Production Start Date: 03/11/03
>
> Version 4.2 Date: 06/01/04
>
> Author: Damon Tighe, Tijana Glavina
>
> Reviewed/Revised by: Lena Philip

# Summary

Upon receiving transformation stocks from the Library Creation group,
the transformation stocks are plated onto 254 x 254mm dry bioassay agar
plates using glass beads. The plates are then incubated for 18 hours in
preparation for colony picking on the QPix instruments.

# Materials & Reagents

+----------------------------+-----------------------+-----------------+
| **[Materials/Reagent       | **[                   | **[Stock        |
| s/Equipment]{.underline}** | Vendor]{.underline}** | Number          |
|                            |                       | ]{.underline}** |
| *                          | Fisher                |                 |
| [Disposables]{.underline}* |                       | 11312D          |
|                            | Corning/VWR           |                 |
| 6mm Glass Beads, Soda Lime |                       | 430052          |
|                            | Corning/VWR           |                 |
| 15-ml Centrifuge Tube      |                       | 430290          |
|                            | Falcon                |                 |
| 50-ml Centrifuge Tube      |                       | 357551          |
|                            | Eppendorf             |                 |
| 10-ml Serological Pipet    |                       | 22363395        |
|                            | Teknova               |                 |
| Safe-Lock 2.0-ml           |                       | L4902           |
| Microcentrifuge Tubes      | Teknova               |                 |
|                            |                       | L4415           |
| *[Reagents]{.underline}*   | Teknova               |                 |
|                            |                       | L4908           |
| LB Amp 100 X-gal plates    | Teknova               |                 |
| (254mm)                    |                       | L4013           |
|                            | Teknova               |                 |
| LB Chlor 20 X-gal plates   |                       | S1625           |
| (254mm)                    | Monahan Paper Company |                 |
|                            |                       | 775011          |
| LB Chlor 12.5 X-gal plates | AAPER Alcohol (LBNL)  |                 |
| (254mm)                    |                       | 6810-13289      |
|                            | Millipore Milli-Q     |                 |
| LB Kan 30 X-gal plates     | System                | \-              |
| (254mm)                    |                       |                 |
|                            | Rainin                | \-              |
| SOC medium, 20ml/vial      |                       |                 |
|                            | Eppendorf             | \-              |
| Pure Bright Germicidal     |                       |                 |
| Bleach                     |                       |                 |
|                            |                       |                 |
| 100% Ethanol (200 Proof)   |                       |                 |
|                            |                       |                 |
| Milli-Q Water              |                       |                 |
|                            |                       |                 |
| *[Equipment]{.underline}*  |                       |                 |
|                            |                       |                 |
| EDP3 Electronic Pipette    |                       |                 |
|                            |                       |                 |
| Easypet                    |                       |                 |
+----------------------------+-----------------------+-----------------+

# Procedure

NOTE 1: Your supervisor will notify you by email on which Libraries you
will be plating.

NOTE 2: Always clean surfaces with 70% ethanol before placing the
bioassays on them.

**1. [Using the Database to Select Libraries]{.underline}**

1.  In the Venonat database, click on **Libraries DB**  select **Choose
    Plating Queue** (Step 4-Plating- in Library Support Database)
    select what type of Library you will be plating (i.e. *Whole genome,
    Evolutionary genomics, All others*)  click on **SUBMIT**.

2.  Upon submitting the queue, a new window titled **Select Libraries
    for Plating** will open that contains all of the libraries possible
    to plate in that Library type (*Refer to Figure 1 in Appendix A*).

3.  You will select which libraries to plate and how many based upon the
    number of 384-well plates that are needed by each library.
    Generally, each bioassay produces two and a half 384-well
    destination plates.

4.  As you select what libraries you are going to plate, fill in your
    initials and the number of barcodes needed (corresponding to the
    number of bioassays).

5.  Once you have a sum of 20 -- 40 bioassays worth of libraries to
    plate, highlight your library selections using the cursor  go to
    **FILE  PRINT  SELECTION  PRINT**.

> ***NOTE:** This page will be helpful as you search for your libraries
> in the freezer as well as with the following steps. Put this copy in
> your lab notebook.*

6.  Click on **SUBMIT**.

> ***NOTE:** Print this page. This page will give you the following
> information: library name, antibiotic, vector, total plates needed,
> total plates created, additional plates needed, total suspension
> volume, plating stock volume and SOC volume.*

7.  The next screen will have a link to print off your barcodes, select
    this link. On the barcode printing page, select SATO 4 as your
    barcode printer and submit (SATO 4 is on counter I of Rm139). Put
    the barcodes on your lab bench, you will be affixing them to dry
    bioassays later.

> **NOTE:** If the barcodes did not already print, press the **Online**
> button of the machine. 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 pressing **Feed**, the
> printer will print multiple copies of your last barcode. The barcodes
> should have the library name and bioassay number on them.

**2. [Drying Bioassays]{.underline}**

> ***NOTE:** When transporting the bioassays on a cart, make sure to
> wipe the cart down with a 70% ethanol and a paper towel.*

1.  Remove the desired number of sleeves of bioassays containing the
    correct antibiotic from the 4^o^C deli.

> ***NOTE:** For **pUC18** and **pMCL200** libraries, plate 1 bioassay
> per 2.5 destination plates needed. For example, if a library needs 6
> destination plates, you will only need to plate 3 bioassays (approx.
> 1:2.5 ratio). For **pcc1Fos** libraries, plate 1 bioassay per
> destination plate (1:1 ratio).*

2.  Check expiration dates on each sleeve. Use the oldest ones first. Do
    not use anything that has expired.

3.  Wearing gloves, slice open each sleeve on a clean workspace.

4.  Examine plates for contamination and irregularities (Irregularities:
    agar cracked, uneven agar levels, too much or too little agar on
    plate etc.). *Refer to Figure 2 in Appendix A.* If the plates are
    unusable, fail the plates out of the system and pass them to QC for
    further testing.

> ***NOTE:** To fail the plates by lot number go to **Venonat 
> Libraries DB  Enter Failed/Not Used Bioassay Barcodes and Lots.**
> Enter in operator, failure mode, lot #, and additional comments. Bring
> the bioassay to QC once it has been failed.*

5.  Dry the outside of the plates with a paper towel and tilt the plates
    on their side and drain any excess liquid from the plate into the
    trash.

6.  Place the plates with their lids down and ajar in the three "Drying
    Only" 37^o^C incubators located in Rms139 and 146, so that as the
    plate warms, moisture can escape.

> ***Important!** Do not place any of the plates against the back of the
> incubator where hot air is blown out of vents. Placing plates in the
> back will cause them to dry non-uniformly and can affect the flatness
> of the agar surface making them difficult for the QPix machines to
> image.*
>
> ***NOTE 1:** If you use* LB Chlor 20 X-gal plates and/or LB Chlor 12.5
> X-gal plates, label the sides of the bioassays in order to
> differentiate between the two different types.
>
> ***NOTE 2:** The bioassays should be stacked 3 per shelf and 13-14 per
> incubator.*

7.  Check the incubators every 15 minutes to see which bioassay plates
    are dry (plates dry at different rates, based on their location in
    the incubator).

> ***NOTE:** Drying time can take approximately 1-2 hours.*

8.  Take out all of the plates that do not contain droplets of moisture
    on the agar surface. If any plates still have liquid on their
    surface, leave them in the incubator for further drying.

> **Important!** The bioassays you plate must be completely dry,
> otherwise the extra moisture will prevent the proper absorption of the
> plating mix and make the plates unpickable. Be careful not to over dry
> the plates - over drying may interfere with proper spreading of
> colonies as well. Examine each bioassay carefully for irregularities
> or contamination before use.

9.  Once you have all of your bioassays, stack them into sets of 20 with
    their lids down.

10. Place the plates upside down with the antibiotic label facing you.
    Affix the barcodes to the right side of the plate.

**3. [Finding your transformation stock]{.underline}**

> ***NOTE 1:** The following steps can be performed while the bioassays
> are drying.*
>
> ***NOTE 2:** If more than one transformation stock is needed, the
> stocks must come from the same transformation event and date.*

1.  Transformation stocks are found in the -80^o^C freezer at the end of
    bay BC in Rm139. Bring an insulated bucket full of ice in which to
    put your transformation stocks.

2.  Using the *Select Libraries for Plating* sheet printed out in step
    **1.2** and the column labeled *Location*, look for the box
    containing your transformation stock. Inside each box are multiple
    transformation stocks with their library name on the cap and you
    will need to hunt through them to find your libraries:

> ***NOTE:** It is easier to pull out a rack and set it on the
> countertop than to shuffle through the freezer with the door open.
> Sometimes the boxes get stuck in the racks and need to be tapped out
> using a blunt tool.*

a.  "Human" and "All Others" libraries can be found in racks labeled
    with an appropriate number only.

b.  "Microbial" libraries can be found in racks labeled with the letter
    "M".

c.  "Whole genomes" are in racks labeled "WG".

```{=html}
<!-- -->
```
3.  As you remove your transformation stocks from their boxes,
    immediately place them on ice.

> ***NOTE:** Transformation stocks will take approximately 15-30 minutes
> to thaw on ice. You can periodically roll the tube in your hand to
> thaw and mix it.*

4.  If you cannot find your transformation stock, go back and repeat
    section **1** to make up the number of bioassays you would have
    plated for the missing stock. Also, alert your supervisor about the
    missing transformation stock.

**4. [Plating Using Glass Beads]{.underline}**

> ***NOTE 1:** Most of the time the plater will plate one whole genome
> library at a time in order to prevent any potential mix ups of
> different plating stocks. In the event of plating more then one whole
> genome library in a plating event, the plater needs to separate all of
> the preparation of the plating stock as well as the actual plating of
> the library.*
>
> ***NOTE 2:** For BAC libraries, the plater can process a maximum of 10
> libraries at a time.*

1.  Preparing the plating mix:

    a.  For libraries that require one bioassay plated you will need to
        use 2-ml tubes, "shooters", for each sample.

    b.  You will need to plate 2ml of the plating mix (SOC media plus
        transformation plating stock) onto each bioassay. *Refer to the
        Preparing Mix Calculations Template* to determine the
        transformation and SOC media volumes needed for the plating mix.
        These are general guidelines you should follow:

        i.  *If the plating volume is less than 10μl*, you can add the
            plating volume on top of the 2ml of SOC for each bioassay
            (no need to subtract).

        ii. *If plating 5 or more bioassays per library and the plating
            volume is larger than 10μl*, subtract the plating volume
            from 2ml to calculate out how much SOC to add to your mix.

        iii. *If the plating volume is 100μl or greater*, you should
             always subtract it from 2ml of SOC per plate.

        iv. For libraries requiring multiple bioassays, you may set up
            your plating mix in a 15-ml or 50-ml centrifuge tube or in a
            small glass bottle (100-ml), depending on the number of
            bioassays you need to plate. Using a measuring cylinder, add
            the total volume of SOC broth needed to plate all the
            bioassays for the particular library.

    c.  Using barrier tips, aliquot the recommended plating volume,
        found on the sheet printed from step **1.7** (*Select Libraries
        for Plating*), into your plating mix tubes and immediately place
        the original stocks back into the -80^o^C freezer.

    d.  Cap the tubes or bottle and label the cap with the library name
        of the stock.

    e.  Mix by gently inverting the tube several times and place it on
        ice.

    f.  Discard any leftover SOC broth. Use a fresh bottle of SOC daily.

2.  Make a collection container for your dirty beads by pouring
    **250ml** of 10% bleach into a 500-ml beaker.

> ***NOTE:** More than 250ml may be needed to completely submerge the
> beads in the 10% bleach solution.*

3.  Set up the hood area where you will be plating (*Refer to Figure 3
    in Appendix A*):

    a.  Spray and wipe down the hood down with 10% bleach and then 70%
        ethanol.

    b.  Put your two stacks of dry plates in the far left corner, your
        dirty bead beaker in the left front corner along with a 500-ml
        beaker to put your empty "shooters" centrifuge tubes in and your
        250-ml beaker full of clean glass beads.

    c.  Transfer your "shooters" from ice to a centrifuge tube rack,
        gently invert the entire rack a few times and place them in the
        front center area of the hood.

4.  Plating with the glass beads:

    a.  Remove 3-4 plates (set 1) from the stack and flip them so their
        lids are up. The number of plates you pull out depends on how
        many you can hold in a stack at one time.

    b.  To each plate, add approximately 10-15 beads.

> ***NOTE:** If too many beads are added by accident, excess beads
> should be dumped into the bleach beaker.*

c.  Mix your shooter or tube containing the plating mix immediately
    before dispensing.

d.  For plating single bioassay per library, lift the first lid ajar and
    dump the contents of the shooter, which corresponds to that bioassay
    (make sure to shake it to get all possible liquid out).

e.  For plating many bioassays of the same library, use a 10-ml pipet to
    draw up 8ml of the mix for 4 bioassays (6ml for 3 bioassays, 4ml for
    2 bioassays, 2ml for 1 bioassay), dispensing **2ml** of the plating
    mix onto each bioassay.

> ***NOTE:** Pour any leftover plating mix onto the last bioassay for
> that particular library.*

f.  Close the lid of the plate and set it to your right with the lid
    facing up. Continue this process with the other three plates and
    then pick up the stack of 4 plates (set 1) and shake them for 15
    seconds or until the solution is well distributed over the plate.

> ***NOTE:** If the bioassays are still somewhat wet, the first shake
> might take up to a minute.*
>
> ***Important!** The shaking should be done thoroughly yet gently. When
> shaking, you should make as many **irregular but gentle** motions as
> possible to ensure that the beads are not taking the same path across
> the plate, otherwise the distribution of colonies will be poor. Make
> sure your feet are firmly on the ground and that you are holding the
> stack of bioassays close to your body while you shake it. You can also
> turn your back towards the hood door and lean on it slightly to gain
> back support. This is to prevent a back injury that may result from
> improper posture while plating.*

g.  Place the stack of 4 plates (set 1) to the far right side of the
    hood and begin adding beads to another stack of 4 plates (set 2).

h.  Once all of the beads have been added to set 2, return to set 1 and
    shake the plates again for 15 seconds or until the plate no longer
    looks *wet*. If the bioassay appears dry after the 1^st^ shake, you
    can do only a brief 2^nd^ shake.

i.  Add your "shooters" to set 2 and shake for 15 seconds or until the
    solution is well distributed over the plate. Place the plates to the
    side.

j.  ![](media/image1.png){width="0.375in"
    height="0.3020833333333333in"}Take each plate in set 1 and
    individually shake it briefly (2 seconds) and then turn it
    vertically with one edge down over the dirty bead beaker and open
    the lid letting all of the beads fall into the beaker.

> ***CAUTION!** Be careful not to splash bleach onto the plate.*

k.  Place the beadless plates in the far right corner of the hood with
    the bioassays facing right side up. Finish the other three plates of
    set 1 and then move on to shaking set 2.

l.  Grab another set of four (set 3) and begin to add the beads.

m.  Continue with this process until you have plated transformation
    stocks onto all of the bioassays and you have a large stack of
    finished plates on your right.

> ***NOTE:** Sometimes, some bioassays are particularly wet and have to
> sit in the hood for a long time before beads can be removed. You can
> leave those ones sitting on beads a few minutes longer and discard the
> beads when the bioassay appears dry.*

5.  Once all of the plates have been beaded, stack them upside down with
    their lids ajar and in a staggered fashion in the hood to let them
    dry. This drying should take about 15 minutes.

6.  During the 15 minutes, you can empty the waste beaker and clean up
    other miscellaneous items. After 15 minutes, check the surface of
    each plate for visible drops of liquid. If all of the plates are
    dry, close lids and leave the plates in a stack with their lids
    facing down.

**5. [Cleanup]{.underline}**

1.  The centrifuge tube and 10-ml pipet tips used for plating should be
    placed in the autoclave waste.

2.  Clean the hood with 10% bleach followed by 70% ethanol.

3.  Cleaning the Beads (only when the bead waste container is full):

    a.  Place parafilm over the top of the beaker and shake the beads in
        the bleach for 10 seconds.

    b.  Empty the bleach into the sink.

    c.  Fill the beaker with tap water, empty, and repeat two more
        times.

    d.  Fill the beaker with Milli-Q water once, shake, and empty into
        the sink.

    e.  Fill the beaker with 70% ethanol, shake, and empty into another
        beaker. Repeat this step one more time.

> ***NOTE:** Do not pour the ethanol into the sink. Pour the ethanol
> into another beaker, so it will be easy to carry to the hazardous
> waste container. Let the leftover ethanol evaporate off of the beads
> in the hood for 10 minutes.*

f.  Dispose of the ethanol in the red hazardous waste container.

g.  Let the beads dry in the fume hood.

h.  Place foil on top of the beaker and give it to autoclaving in Rm143.

i.  When beads have been autoclaved, pour into bead waste container for
    glass recycling.

**6. [Incubation and "In Plating" Database]{.underline}**

1.  Place the bioassays upside down in stacks of five into one of the
    "incubation only" 37^o^C incubators for 18 hours. Place a maximum of
    five stacks of five plates per shelf of the incubator. On the very
    top shelf, place only two stacks of five plates.

2.  Record *your initials, the number of bioassays, the date, and the
    time in, and the time out* (18 hours later) on the Bioassay
    Incubation sheet on the outside of the incubator

> ***NOTE:** 18 hours is an approximation since colonies generally grow
> in 18 hours but some will require more or less time in the incubator
> to reach adequate size. The pickers will make sure the colonies are of
> good size before the plates are pulled out of the incubators.*

3.  In the Venonat database, click on **Libraries DB**  select **In
    Plating**  on the next page are rows that tell you information
    about what you have plated, including how many bioassays you have
    made and the number of destination 384-well plates that were
    requested.

4.  You will need to fill out the **Next Step** column on libraries you
    have plate using the drop down list in each cell.

> ***NOTE:** The 4 dropdown menu options are Plating Complete -- Stock
> Left, Plating Complete -- No Stock Left, Some Plating -- Return for
> More Plating, Did not plate -- Return for plating.*

5.  If you select *Plating Complete -- No Stock Left*, notify your
    supervisor that the stock is empty.

6.  Click on **SUBMIT**.

7.  The next day after plating, the pickers will remove the plates from
    the incubator and put them in the deli of Rm147, where unpicked
    plates are kept. You will need to examine your plates and look for
    anything irregular, such as large colonies, discolored colonies, and
    colony smearing due to liquid. Make note of any irregularities in
    your notebook and anything that may have caused them. Alert the
    supervisor, if necessary.

> ***NOTE:** If the plates are unusable, fail the plates out of the
> system. To fail the plates with barcodes go to **Venonat  Libraries
> DB  Enter Failed/Not Used Bioassay Barcodes and Lots.** Enter in
> operator, failure mode, lot #, barcode, and additional comments. Bring
> the bioassay to QC once it has been failed.*

8.  Each plater is responsible to keep track of their colony counts
    daily and keep track of them in their notebook. Report to supervisor
    if you notice any irregularities.

# Reagent/Stock Preparation

**[10% Bleach]{.underline}**

In a 2-L bottle: Add **200ml** bleach to **1800ml** Milli-Q water. Mix
well. Label: *10% Bleach, date,* and *your initials*.

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

In a 100-ml bottle, add **70ml** 100% ethanol to **30ml** Milli-Q water.
Mix well. Label: *70% Ethanol, date,* and *your initials*.

# Troubleshooting

If, after the barcodes have already been printed, you notice that there
is a contaminated bioassay or you will be unable to finish plating the
transformation stocks for that day, go to the Venonat database, click on
**Libraries DB**  select **Scan Bioassays Failed or Unused**  scan in
the failed or unused barcodes.

# 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

Figure 1. **Select Libraries for Plating Window**. Each row describes a
different library. The rows are color coded to tell you what type of
antibiotic each library needs to be plated on. In the top left corner of
the window is the key for antibiotic and row colors.

![](media/image2.png){width="6.5in" height="3.338188976377953in"}

Figure 2. Examining bioassays for contamination or irregularities.

![](media/image3.jpeg){width="2.2818897637795277in"
height="1.7271653543307086in"}![](media/image4.jpeg){width="2.0535433070866143in"
height="1.706299212598425in"}![](media/image5.jpeg){width="2.358267716535433in"
height="1.8854330708661418in"}![](media/image6.png){width="2.206299212598425in"
height="1.8515748031496062in"}

![](media/image7.jpeg){width="2.358267716535433in"
height="1.7811023622047244in"}![](media/image8.png){width="2.205511811023622in"
height="1.7244094488188977in"}

Figure 3. Fume hood setup for plating with glass beads.

# Appendix B

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

**5/26/04** -- Christine Lansang and Chris Daum performed an audit
between 9:15AM -- 1:40PM. The user was Miranda Harmon-Smith. Changes
incorporated into protocol on **8/25/04**.