Large Clone Maxi-Prep

> Version Number: 3.4
>
> Production Start Date: 01/02/02
>
> Version 3.4 Date: 06/09/04
>
> Authors: Miranda Harmon-Smith, Anna Ustaszewska
>
> Reviewed/Revised by: Tijana Glavina

# Summary

The QIAGEN Maxi-Prep kit is used to isolate BAC, fosmid or cosmid DNA
from glycerol stocks of clones submitted to the JGI. *NOTE: This
protocol does not contain an exonuclease to remove the E. Coli
contamination.*

# Materials & Reagents

+----------------------------+-----------------------+-----------------+
| **[Materials/Reagent       | **[                   | **[Stock        |
| s/Equipment]{.underline}** | Vendor]{.underline}** | Number          |
|                            |                       | ]{.underline}** |
| *                          | Falcon                |                 |
| [Disposables]{.underline}* |                       | 357550          |
|                            | Falcon                |                 |
| 50-ml Serological Pipet    |                       | 357525          |
|                            | Falcon                |                 |
| 25-ml Serological Pipet    |                       | 357551          |
|                            | Falcon                |                 |
| 10-ml Serological Pipet    |                       | 357543          |
|                            | \-                    |                 |
| 5-ml Serological Pipet     |                       | \-              |
|                            | \-                    |                 |
| Inoculation Loop           |                       | \-              |
|                            | QIAGEN                |                 |
| 1-ml Cryovial-Nunc         |                       | 12163           |
|                            | Teknova               |                 |
| *[Reagents]{.underline}*   |                       | L8000           |
|                            | Ambion                |                 |
| QIAGEN Plasmid Maxi Kit    |                       | 9849            |
| (25):                      | Acros                 |                 |
|                            |                       | 67-63-0         |
| (QIAGEN-tip 500, RNase A   | AAPER Alcohol (LBNL)  |                 |
| (100mg/ml), P1 Buffer, P2  |                       | 6810-13289      |
| Buffer, P3 Buffer,         | Aldrich               |                 |
|                            |                       | 857440-25G      |
| QBT Buffer, QC Buffer, QF  | USB                   |                 |
| Buffer)                    |                       | 25389-94-0      |
|                            | Sigma-Aldrich         |                 |
| LB Broth                   |                       | L4522           |
|                            | J.T. Baker            |                 |
| TE Buffer, pH 8.0          |                       | 1310-73-2       |
|                            | Monahan Paper Company |                 |
| 99.9% Isopropanol, HPLC    |                       | 775011          |
| Grade                      | Sigma                 |                 |
|                            |                       | G5516-1L        |
| 100% Ethanol (200 Proof)   | Millipore Milli-Q     |                 |
|                            | System                | \-              |
| Chloramphenicol (98%)      |                       |                 |
|                            | **[                   | **[Stock        |
| Kanamycin Sulfate          | Vendor]{.underline}** | Number          |
|                            |                       | ]{.underline}** |
| Sodium Dodecyl Sulfate     | \-                    |                 |
| (SDS)                      |                       | \-              |
|                            | \-                    |                 |
| 1N Sodium Hydroxide        |                       | \-              |
|                            | Sorvall/Beckman       |                 |
| Pure Bright Germicidal     |                       | \-              |
| Bleach                     | Sorvall/Beckman       |                 |
|                            |                       | \-              |
| Glycerol, for molecular    | Innova                |                 |
| biology, 99%               |                       | \-              |
|                            | Eppendorf             |                 |
| Milli-Q Water              |                       | \-              |
|                            | \-                    |                 |
| **[Materials/Reagent       |                       | \-              |
| s/Equipment]{.underline}** |                       |                 |
|                            |                       |                 |
| *[Equipment]{.underline}*  |                       |                 |
|                            |                       |                 |
| 250-ml Centrifuge Bottles  |                       |                 |
|                            |                       |                 |
| 50-ml Centrifuge Bottles   |                       |                 |
|                            |                       |                 |
| Sorvall/Beckman            |                       |                 |
| Centrifuges                |                       |                 |
|                            |                       |                 |
| Rotors (SLA 1500, JA 14,   |                       |                 |
| SA 600, JA 17)             |                       |                 |
|                            |                       |                 |
| 4900 Upright Shaker        |                       |                 |
|                            |                       |                 |
| Easypet                    |                       |                 |
|                            |                       |                 |
| Precision 180 Series water |                       |                 |
| bath                       |                       |                 |
+----------------------------+-----------------------+-----------------+

# Procedure

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

**1. [Entering Clones into the Isolation Queue]{.underline}** (Do when
time permits -- day prior to or day of prep).

1.  Go the **Isolations** Excel file in the following directory:
    [**\\\\Octopus\\ProdSeq\\Lib\\Libraries
    Shared**](../../../../../../../../ProdSeq/Lib/Libraries%20Shared).

2.  Copy all of the JGI Clone IDs from the appropriate date. Paste into
    notepad.

3.  In the Venonat database
    ([**http://venonat.jgi-psf.org/psf/PSF.home**](http://venonat.jgi-psf.org/psf/PSF.home)),
    click on **Libraries DB**  select **Library Reports**  select
    **Query/Enter Project Form**.

    a.  Paste the Clone ID from step **1.2** into *Project ID* box.

    b.  Click on **SUBMIT**.

4.  Click on the highlighted Library name.

5.  Under the Queue to Process dropdown menu, select **Ready to
    Isolate**.

6.  Double check to make sure that the right vector, insert size, etc.
    information is listed.

7.  Click on **SUBMIT** (sent to Isolations Queue).

8.  Select and copy the next Clone ID from the Isolations notepad file,
    and repeat steps **1.3 --1.7**. Continue until all of the clones
    have been entered and sent to the Isolations Queue.

9.  Check the Isolation Queue in Venonat database to make sure all
    libraries have been entered

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

***NOTE:** The day prior to inoculation, incoming glycerol stocks or
stabs should be streaked onto a small agar plate containing the
appropriate antibiotic to ensure inoculation from a single colony of
growth. Incubate the plate at 37°C for 16-18 hours or until the colonies
are large enough from which to pick. Place streaked clones in the 4°C
deli until ready for inoculation. Glycerol stocks coming from LLNL do
not need to be streaked since they were picked from a single colony
prior to shipping. Used streaked plates and stab cultures are kept in
the deli for up to 1 month before being discarded in autoclave waste.*

1.  Pull the streaked clones from the 4°C deli. If you are inoculating
    from a glycerol stock, pull a designated clone for inoculation out
    of the freezer to thaw on ice. If any of the clones are cosmids,
    remove the Kanamycin antibiotic from the freezer so that it can thaw
    at room temperature.

2.  Label twenty-five 500-ml flasks *1-25* with tape. Record in your lab
    notebook which clones (use JGI ID's) are designated 1-25.

3.  Measure **250ml** of LB Broth in a graduated cylinder and then pour
    into each flask. Record the LB lot \# in your notebook or on the
    Isolations Excel spreadsheet. Use the same LB lot for all clones
    being isolated, if possible.

4.  Remove the appropriate antibiotic from the small freezer in room 139
    and add the correct amount to each flask. Tip the flask so the tip
    of the pipette touches the LB broth and dispense the antibiotic.

    a.  For BAC or fosmid clones: add **110µl** of 34mg/ml of
        Chloramphenicol per sample flask. Final concentration = 15μg/ml.

    b.  For cosmid clones: add **313µl** of 20mg/ml of Kanamycin per
        sample flask. Final concentration = 25μg/ml.

5.  Inoculate each flask with a single colony from a streaked plate (use
    a sterile pipette tip or an inoculating loop) or **12µl** of
    glycerol stock. Tilt the flask to avoid touching the pipette to the
    inside of the flask. Only the tip should touch the growth medium.

6.  Grow culture flasks at 37 °C for **18 -- 20 hours at 200rpm** in the
    Innova 4900 upright shaker (Rm146). Press the Start button and wait
    to make sure the shaker is running properly.

**3. [Prep for next day:]{.underline}**

1.  Make sure that all the plastic ware you will be using is autoclaved.
    You will need for each sample: *one 250-ml centrifuge bottle and
    four 50-ml centrifuge tubes*. If you plan to inoculate again
    tomorrow, make sure you will have enough 500-ml flasks.

2.  Make sure you have enough Qiagen reagents and columns. For the
    isolation of 25 samples, use an entire QIAGEN kit. If there are
    remaining reagents due to a small sample batch, label the bottles
    and kit with the date, your initials, and approximately how much
    buffer is left: i.e. enough for 10 clones. Do not use left over
    reagents if more than a week has passed.

3.  Add RNase A (entire tube, approximately 280ul) to P1 buffer and mix
    by inversion. Place the P1 buffer and the P3 buffer in the deli at
    4°C for use the next day.

4.  Label the 250-ml centrifuge bottles (1-25) with tape and 4 sets of
    twenty-five 50-ml centrifuge tubes 1-25 with a sharpie. *Check the
    condition of the tubes for debris. If debris is present do not use.*
    Put the 50-ml tubes in racks and cover the tops of the tubes with a
    paper towel so that nothing falls into them overnight.

5.  Label 25 cryovial tubes with a "fat label" and 1-25 on the top if
    making glycerol stock tubes the next day.

6.  Creating labels for glycerol stock tubes and 2 sets of Eppendorf
    tubes for isolated DNA:

> ***NOTE:** Glycerol stocks should be relabeled around the tube with a
> "fat label" containing the JGI ID number and Source I.D. name and a
> round label on top with the library name. Glycerol stocks are stored
> in the Glycerol Stock Freezer for future use.*

a.  Go the **Isolations** file in the following directory:

> [**\\\\Octopus\\ProdSeq\\Lib\\Libraries
> Shared**](../../../../../../../../ProdSeq/Lib/Libraries%20Shared)

b.  Copy the JGI Clone ID and source clone name columns that contain the
    samples that are to be quantified.

c.  Paste the JGI Clone ID and clone name columns into notepad.

d.  Go to the **Fat Labels** document in
    [**\\\\Octopus\\ProdSeq\\Lib\\Libraries
    Shared**](../../../../../../../../ProdSeq/Lib/Libraries%20Shared).

e.  From notepad, copy and paste the ID and clone name into each fat
    label. Do this in triplicate as you will need one label for the
    glycerol stock vial, DNA for shearing tube, and DNA isolation tube.

f.  Print on the label paper.

g.  Place the labels on 1.5-ml Eppendorf tubes (2 sets) and glycerol
    stock tubes (cryovials).

h.  Prepare round labels for on top of each tube with the library name.

### **4. [Isolation-modified Qiagen Large Construct protocol]{.underline}**

> ***NOTE**: Be careful during transfer steps not to mix up the clones.
> Also, if making glycerol stocks for each clone, aliquot 300μl of 30%
> glycerol into each labeled cryovial prior to adding cell culture
> aliquot.*

1.  Remove culture flasks from shaker incubator. Pour each culture into
    its corresponding 250-ml centrifuge bottle. Check for washer in the
    cap, w/o --leaking occurs. If making a glycerol stock, remove 600μl
    aliquot and place in labeled cryovial tube containing 300ul 30%
    glycerol. Tighten caps well. Place the glycerol stock tubes in the
    --80^o^C freezer after labeling the tops with the round library name
    labels.

2.  Pellet the cells by spinning the 250-ml centrifuge bottle down at
    4°C for **15 minutes at 4,500 rpm**. Clean out water in centrifuge
    if present (may cause off-balance). You can load all 4 centrifuges
    at once being sure appropriate rotors are in.

> ***NOTE 1:** Make sure the appropriate rotors are in the centrifuges
> depending on which centrifuge you will be using and whether you will
> be spinning the 250-ml bottles or 50-ml tubes.*
>
> ***NOTE 2:** While spinning clean used flasks-, remove tape, rinse the
> flasks with water in the sink and put in the autoclave bin. Get ice in
> ice bucket for subsequent step and place 1 set of 25 tubes into the
> ice.*

3.  Turn on water bath to heat to 65^o^C. Place elution buffer (QF) in
    water with lid slightly opened.

> ***NOTE:** This buffer needs to be heated to 65^o^C to better elute
> the DNA from the column.*

4.  After the centrifuge spin, decant the supernatant into a waste
    beaker. Pour the supernatant away from the pellet -- invert the
    bottle on a paper towel to drain.

    a.  Add 5% hypochlorite solution (bleach) to the supernatant to kill
        leftover bacterial cells. The final concentration of the
        hypochlorite should be 0.5%.

> ***NOTE:** Usually we collect 4L of supernatant at once, so you need
> to add **400ml** of 5% hypochlorite solution to end up with 0.5%
> solution. If you have any other volume of the supernatant, you need to
> add appropriate amount of the 5% hypochlorite to achieve the final
> concentration needed.*

b.  Dump the waste down the sink after 10-15 minutes and wash it down
    the sink with cold water.

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5.  Resuspend the cell pellet in **10ml** of P1 (Resuspension) buffer.
    Make sure RNase has been added (Refer to **Reagent/Stock
    Preparation**). To resuspend:

    a.  Use the Easypet and a sterile 10-ml pipet to squirt the P1
        buffer into the bottle.

    b.  Suck approximately 5mls back into the pipet and squirt it at the
        pellet again. Repeat several times until there are no visible
        cell clumps or cells attached to the wall of the bottle.

    c.  Discard the pipet in the autoclave waste container and use a new
        pipet for each sample.

6.  Set one timer for 5 minutes and another for 15 minutes but do not
    start the timers. Obtain the P2 buffer from the kit and check for
    precipitation. If precipitate, warm to 37^o^C to resuspend.

7.  Gently add **10ml** of P2 (lysis) buffer to the resuspended cells by
    gently letting it drip down the side of the bottle. As soon as you
    add the P2 buffer to the first bottle, start the 5-minute timer. Do
    not mix. Obtain the P3 buffer from the deli for the next step.

> ![](media/image1.png){width="0.375in"
> height="0.3020833333333333in"}***Important!** Do **not** shake the
> centrifuge bottle. Vigorous shaking at this step could lead to
> shearing of the bacterial genomic DNA and make it impossible to
> separate BAC from genomic DNA. P2 Buffer should be closed immediately
> after use to avoid acidification from CO~2~ in the air.*

8.  At the end of 5 minutes, gently add **10ml** of chilled P3
    (Neutralization) buffer to the lysed material. As soon as you add
    the P3 buffer to the first bottle, start the 15-minute timer. Do not
    mix.

> ***Important!** The cells should lyse for no longer than 5 minutes.
> Sufficient mixing occurs when buffer is introduced to the solution. No
> separate mixing is needed.*

9.  Immediately after all 25 bottles have the P3 added, pour the
    contents of the 250-ml centrifuge bottles into 50-ml centrifuge
    bottles being careful to minimize shaking. Put on ice until the
    15-minute neutralization step is completed.

> ***NOTE:** Steps **4.8** and **4.9** should all occur within the
> 15-minute neutralization period.*

10. Place caps on the 50-ml tubes immediately after pouring contents
    into the tubes. Again, be careful not to disturb the contents.

11. Spin 1: At the end of the 15-minute neutralization period,
    centrifuge the samples at 4°C for **30 minutes at 15,000rpm**. The
    BAC DNA will remain in solution; the genomic DNA, proteins, cell
    debris, and SDS will form a pellet. Be sure to load the tubes in the
    centrifuge with the numbers facing out. Also make sure correct rotor
    is in place for the 50-ml tubes.

12. While spinning:

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a.  Clean the used 250-ml centrifuge bottles by removing tape, filling
    with warm water, and scrubbing with bottlebrush. Rinse with water
    and place in the gray wash bins.

b.  Discard the ice.

    1.  Spin 2: After the 30-minute spin is complete, gently transfer
        the supernatant to a clean 50-ml centrifuge tube and spin at 4°C
        for **15 minutes at 15,000rpm**. It is important to do this step
        gently in order to minimize the transfer of waste materials into
        the clean tube. Pour away from the pellet to avoid dislodging.
        You can re-use the same lids being sure to keep the same lid
        with the same sample throughout. i.e. the lid from tube 1 always
        gets put on tube 1.

    2.  While spinning:

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a.  Set up the batch of Qiagen columns using the blue racks. Each rack
    holds four columns so you will need 7 racks for a batch of 25. Place
    50-ml waste tubes in the bottom and new columns in the top of the
    rack. Label each column 1-25 with a sharpie. Equilibrate the columns
    with **10ml** of QBT (Equilibration) buffer. Allow the buffer to
    empty by gravity flow.

b.  Wash the dirty centrifuge tubes from the first spin. Remove the
    number on the outside with 100% ethanol. In the sink, fill the tubes
    with water and use the bottlebrushes to scrub them out. Rinse with
    hot water and place in the gray wash bins.

    1.  Spin 3: After the 15-minute spin is complete, transfer the
        supernatant to a clean 50-ml centrifuge tube and spin one last
        time at 4°C for **15 minutes at 15,000rpm**. Again, pour away
        from the pellet. Re-use the same lids. Put the tubes in the
        centrifuge with numbers facing out.

    2.  While spinning: Wash the dirty centrifuge bottles from spin 2 as
        previously done.

    3.  After the final spin is complete, immediately pour the entire
        supernatant gently into the equilibrated column and allow it to
        empty by gravity flow. Try to minimize disturbance of the
        precipitate as it could clog the columns and pour away from the
        pellet.

    4.  Wash the columns 2 times with **30ml** (fill to the top of
        column) aliquots of QC (Wash) buffer. Allow emptying by gravity
        flow. You will need to discard the flow-through after each wash
        into a waste beaker because it contains ethanol. The waste will
        later be dumped into the red SAA Hazardous waste carboy under
        the lab bench.

> ***NOTE:** The BAC DNA will bind to the membrane in the QIAGEN-tip and
> all contaminants will be washed through.*

5.  Elute the DNA from the columns into a clean 50-ml centrifuge tube
    with heated (65°C) QF (Elution) buffer: Make sure the tubes are
    straight up/down to prevent spilling. Good alignment.

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a.  Add **8ml** of the heated QF buffer then reheat the buffer bottle in
    water bath while buffer is eluting out the DNA.

b.  Add another **7ml** of reheated QF buffer to finish off the elution.

> ***NOTE:** Because of its large size, BAC DNA elutes most efficiently
> at 65°C. That is why it is important to not let the buffer cool during
> elution*.

c.  Empty remaining QF buffer into the hazardous waste and discard used
    columns into the biohazard waste container.

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6.  Precipitate the DNA by adding **10.5ml** of 100% isopropanol at room
    temperature to each tube. Gently dispense isopropanol down the side
    of the tube. Cap the tubes with new lids (make sure there is a seal
    inside the lid). Mix well by inverting the tube 4-5 times.
    Centrifuge at 4°C for **30 minutes at 14,000rpm**.

7.  Carefully decant the supernatant as not to disturb the precipitated
    DNA pellet. The isopropanol pellet will have a glassy appearance
    (invisible) and will be relatively loosely attached to the side of
    the bottle. Pour away from the pellet, which should be on the
    numbered side of the bottle. After decanting  place tube back up
    right in rack  save caps in order to re-use them for the next step.

8.  Wash the DNA pellet:

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a.  Add **5ml** of 70% ethanol to the pellet (make fresh daily \~125ml).
    Dispense ethanol away from the pellet, down opposite side of tube
    from which the pellet is on.

b.  Spin down at 4°C for **15 minutes at 14,000rpm**.

> ***NOTE:** The ethanol pellet may be visible as a flocculent white
> material.*

1.  Carefully decant the ethanol away from the pellet without disturbing
    the DNA pellet. Gently place the tube upside-down on a paper towel
    to get rid of excess ethanol and minimize drying time. Leave upside
    down until all are decanted.

2.  Air dry the pellet in a vent hood for 1-3 hour until the majority of
    the ethanol has evaporated. Angle the tubes so that the ethanol does
    not contact the DNA pellet (keep numbered side facing up).
    Over-drying the pellet will make the DNA more difficult to
    redissolve.

> ***NOTE:** Drying usually takes 3 or more hours until all traces of
> ethanol droplets are gone.*

3.  Resuspend the pellet in **400µl** of TE buffer (pH 8.0). Put new,
    sterile caps on the tubes (tightly) and swirl the buffer around in
    the bottle so that all of the DNA will get resuspended.

> ***NOTE:** If you are processing a sample that had visibly less growth
> following the incubation step, i.e. smaller cell pellet, resuspend DNA
> in only **200μl** of TE buffer. This makes the sample more likely to
> pass the Quantification step later on.*

4.  Leave the tubes to resuspend overnight in a deli refrigerator at
    4°C. Angle the tubes so that the TE completely submerges the DNA
    pellet to ensure that the whole pellet goes back into solution.
    Anchor the tube rack so that the rack does not tip over. Numbers on
    tubes should be facing down when rack is tilted.

5.  In the morning, swirl the buffer up and down the sides of the tube,
    centrifuge the tubes for **1 min at 14,000 rpm**, and transfer the
    contents of the tubes to 1.5-ml Eppendorf tubes labeled on top 1-25
    and on the side with a fat label. Place DNA tubes in DNA stock
    freezer for later use in quantification step, unless using same day
    to quantify then place in deli.

6.  Clean used 50-ml tubes using same procedure as before. Clean off
    numbers from outside and rinse with water. Place in gray wash bins.

**5. [Pass samples in the database to the next step
(Quantification)]{.underline}**

1.  In the Venonat database, click on **Libraries DB**  click on
    **Isolations Queue**  click on **Click here to assign projects to a
    batch**.

2.  Select the operator initials, type in *Large Clone Maxi-Prep* as the
    protocol, and select all of the project IDs that you isolated.

3.  Click on **SUBMIT**.

4.  Click on **Click Here**.

5.  Under the QC dropdown menu, select whether the isolation passed or
    failed (with a given reason).

6.  Select whether a new glycerol stock was made or not.

7.  Enter the source Library name.

8.  Click on **SUBMIT**.

# Reagent/Stock Preparation

**[70% Ethanol (v/v)]{.underline}**

Add **350ml** of 100% ethanol and **150ml** of Milli-Q water to a 1-L
bottle.

Total volume: 500ml.

**[P1 (Resuspension) Buffer]{.underline}**

50mM Tris·Cl, pH 8.0

10mM EDTA

100mg/ml RNase A

**[P2 (Lysis) Buffer]{.underline}**

200mM NaOH

1% SDS (w/v)

If necessary:

Add **262.5ml** of Milli-Q water, **75ml** of 1N NaOH, and **37.5ml** of
SDS to a 500-ml bottle.

Total volume: 370ml

Make fresh the day of the large clone isolation procedure.

**[P3 (Neutralization) Buffer]{.underline}**

3.0M Potassium Acetate, pH 5.5

Make sure P3 buffer is ice cold before starting the large clone
isolation procedure.

**[QBT (Equilibration) Buffer]{.underline}**

750mM NaCl

50mM MOPS, pH 7.0

15% isopropanol (v/v)

0.15% Triton X-100 (v/v)

**[QC (Wash) Buffer]{.underline}**

1.0M NaCl

50mM MOPS, pH 7.0

15% isopropanol (v/v)

**[QF (Elution) Buffer]{.underline}**

1.25M NaCl

50mM Tris·Cl, pH 8.5

15% isopropanol (v/v)

**[LB Broth]{.underline}**

1% Tryptone

0.5 % Yeast Extract

1% Sodium Chloride

**[Chloramphenicol (34 mg/ml) Preparation:]{.underline}**

> Add **0.51g** of chloramphenicol to **15ml** of 100% ethanol to make a
> 34mg/ml chloramphenicol solution.

**[Kanamycin (20 mg/ml) Preparation:]{.underline}**

Add **0.6g** of kanamycin to **30ml** of Milli-Q water to make a 20mg/ml
kanamycin solution.

**[TE Buffer]{.underline}**

10mM Tris, pH 8.0

1mM EDTA, pH 8.0

# 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

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

**6/01/04 -- 6/03/04** -- Danielle Mihalkanin and Erika Lindquist
performed an audit between 7:30PM (6/01) -- 1:30PM (6/03). The user was
Jenny Grant. Changes incorporated into the protocol on **8/26/04**.