### **Library Creation Process SOP:**

### **Large clone Maxi-Prep** 

> Version Number: 3
>
> Start Production Date: 01-02-02
>
> Author: Jason Olivas
>
> Reviewed by: Jenna Morgan/Sanjay Israni
>
> SOP Change Document: x

# Summary

To isolate plasmid DNA from glycerol stocks of clones submitted to the
JGI. This protocol does not contain an exonuclease to remove the e.coli
contamination.

# Materials & Reagents

> **[Materials/Reagents/Equipment]{.underline} [Vendor]{.underline}
> [Stock Number]{.underline}**

##### [Disposables]{.underline}

> 50-ml pipettes Falcon 357550
>
> 25-ml pipettes Falcon 7525
>
> 10-ml pipettes Falcon 357551
>
> 5-ml pipettes Falcon 357543
>
> 1-ml pipettes Fisherbrand 13-678-11b

##### [Stock Solutions]{.underline}

> DDD Water JGI N/A
>
> 70%Ethanol JGI See Below
>
> Tris-EDTA JGI See Below
>
> Chloramphenicol (15ug/ml) Sigma C7795
>
> Kanamycin (25ug/ml) Sigma
>
> RNAseA (100mg/ml) Sigma R6513

##### [Reagents]{.underline}

> Qiagen Maxi-prep Kit Qiagen 12165
>
> SDS (Lauryl Sulfate) Sigma L-4522
>
> 1N NaOH Fisher SS266-1
>
> LB Broth Teknova 0181-iL
>
> 100% Isopropanol VWR PX-1835-14
>
> ATP Teknova

##### [Equipment]{.underline}

> 250 ml Centrifuge bottles Nalgene 3141
>
> 50 ml Centrifuge tubes Nalgene 3139
>
> Sorvall/Beckmen Centrifuges Sorvall/Beckman
>
> Innova 4900 upright shaker

# Procedure

[Inoculation:]{.underline}

1.  Pull clones for inoculation out of the freezer to thaw. If any of
    the clones are cosmids, get the Kanmycin out of the freezer to thaw.

2.  Label 30 500ml flasks 1-30 with tape. Record in your lab notebook
    which clones (use JGI ID's) are designated 1-30. Make sure that it
    is obvious which 30 flasks are yours if someone else is isolating at
    the same time.

3.  Pour 250mls of LB Broth into each flask.

4.  Add appropriate antibiotic: 110 µl of 34 mg/ml of Chloramphenicol
    (15µg/ml) to BAC clones or 313 µl of 20 mg/ml of Kanamycin (25µg/ml)
    to cosmid clones. Both antibiotics are in the small freezer.

5.  Inoculate with 12 µls of glycerol stock for one unique clone. Tilt
    the flask to avoid touching the pipette to the inside of the flask.
    Only the tip should touch the growth medium. If you are inoculating
    from a stab, use an inoculation loop to pick a colony from the stab
    and innoculate the flask. If you cannot see a colony, scrape the
    loop across the surface of the stab and use that.

6.  Grow for 18 hours in the Innova 4900 upright shaker at 37 °C at 200
    rpms.

[Prep for tomorrow:]{.underline}

1.  Make sure all the glassware you will need is autoclaved. You will
    need (for each sample) one 250-ml centrifuge bottle, 4 50-ml tubes.
    If you plan to inoculate again tomorrow, make sure you have enough
    500-ml flasks.

2.  Make sure you have enough Qiagen reagents and columns.

3.  Add RNAse to P1 buffer and chill enough of it and the P3 buffer at
    4°C for use tomorrow.

4.  **If you have time:** label the centrifuge bottles and tubes 1-30.
    You can label the 250-ml bottles with tape, but with the 50-ml tubes
    use a Sharpie, not tape. Cover the tops of the tubes with a paper
    towel so that nothing falls into them overnight. Put the correct
    rotors into the centrifuges.

### [Isolation-modified protocol of the Qiagen Large Construct protocol]{.underline}

**Note: Be careful during transfer steps to not mix up the clones.
Always spin tubes with the numbers facing to the outside.**

1.  Put the correct rotors into the centrifuges.

2.  Check the P2 buffer in the Qiagen kits. If there is a precipitate in
    the bottle, make new lysis buffer according to the recipe.

3.  Turn on the water bath to 65°C.

4.  Label 250-ml centrifuge bottles 1-30. Pour culture into
    corresponding 250-ml centrifuge bottle. Pellet cells at 4,500 rpms
    for 15 minutes at 4°C.

5.  **While spinning:** label 50-ml centrifuge tubes 1-30, rinse out
    flasks, and put the tubes on ice. (Here, you put the tubes deep into
    the ice so that you do not need to do this at a later time when you
    must be gentle with the tubes.)

6.  Decant supernatant into waste beaker. Add about 250ml bleach
    solution to supernatant to kill leftover bacterial cells. Dump waste
    down the sink.

7.  Resuspend cell pellet in 12 mls of P1 (Resuspension) buffer. Make
    sure RNAse has been added. To resuspend, use the Easypet and a
    sterile 10-ml pipette. Squirt the P1 into the bottle, then suck some
    of it back into the pipette and squirt it at the pellet again. Do
    this until there are no cell clumps visible. Use a new pipette for
    each sample. Discard pipettes in the autoclave waste container.

8.  Set one timer for 5 minutes and another for 15 minutes. Get enough
    P2 (or your lysis buffer) and chilled P3 buffer out before beginning
    next step.

9.  Add 12 mls of P2 (lysis) buffer to the resuspended cells. As soon as
    you add the P2 buffer to the first bottle, start the 5-minute timer.
    Although you need to make sure that the lysis buffer is well mixed
    with the P1 buffer, **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. Buffer
    P2 should be closed immediately after use to avoid acidification
    from CO~2~ in the air.

10. At the end of 5 minutes, add 12 mls 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. **The cells should lyse
    for no longer than 5 minutes.** If the P3 is not completely mixed
    with the lysis buffer, some SDS may remain in the supernatant, and
    it will inhibit the binding of DNA to the QUIAGEN-tip membrane.

11. 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. **Steps 9 and
    10 should all occur within the 15-minute neutralization period.**
    Put caps on the bottles now.

12. At the end of the 15-minute neutralization period, centrifuge the
    samples at 15,000 rpms for 30 minutes at 4°C. The BAC DNA will
    remain in solution; the genomic DNA, proteins, cell debris, and SDS
    will form a pellet.

13. **While spinning:** label another group of centrifuge bottles 1-30.
    You do not need new lids for these bottles, just transfer the lid
    from the old bottle. Put the QF buffer into the 65°C water bath. Set
    up the appropriate amount of Qiagen columns and equilibrate them
    with 10mls of QBT (Equilibration) buffer. Allow the buffer to empty
    by gravity flow. Rinse out the 250-ml centrifuge bottles.

14. After the 30-minute spin is completed, transfer the supernatant to a
    clean 50 ml centrifuge bottle and spin at 15,000 rpms for 15 minutes
    at 4°C.

15. **While spinning:** Label another set of 50-ml centrifuge bottles
    1-30. Wash the dirty centrifuge bottles.

16. After the 15-minute spin is completed, transfer the supernatant to a
    clean 50 ml centrifuge bottle and spin one last time at 15,000 rpms
    for 15 minutes at 4°C.

17. **While spinning:** Label another set of 50 ml centrifuge bottles
    1-30. Wash the dirty centrifuge bottles.

18. After the final spin is complete, immediately pour the entire
    supernatant onto the equilibrated column and allow it to empty by
    gravity flow. Try to minimize disturbance of the precipitate as it
    could clog the columns.

19. Wash the columns with two (2)-30ml aliquots of QC (Wash) buffer.
    Allow emptying by gravity flow. You will need to discard the
    flow-through after each wash. The BAC DNA will bind to the membrane
    in the QIAGEN-tip and all contaminants will be washed through.

20. Elute the DNA from the columns into a clean 50-ml centrifuge tube
    with heated (65°C) QF (Elution) buffer. Add 8mls of the heated
    buffer then reheat it while buffer is eluting out the DNA. Add
    another 7mls of reheated buffer to finish off the elution. Because
    of its large size, BAC DNA elutes most efficiently at 65°C. That's
    why it is important not to let the buffer cool during elution.

21. Precipitate the DNA with 10.5mls of 100% room temperature
    isopropanol. Mix and centrifuge at 14,000 rpms for at least 30
    minutes at 4°C.

22. **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.

23. Wash the DNA pellet with 5mls of 70% ethanol by spinning the pellet
    with the ethanol at 14,000 rpms for 15 minutes at 4°C. The ethanol
    pellet may be visible as a fluffy white material.

24. **Carefully** decant the ethanol without disturbing the DNA pellet.
    Gently place the bottle upside-down on a paper towel for a second to
    get rid of excess ethanol and minimize drying time.

25. Air dry the pellet in a vent hood for 1 hour or more until the
    majority of the ethanol has evaporated. Angle the tubes so that the
    ethanol is as far away as possible from the DNA pellet as possible.
    Over-drying the pellet will make the DNA more difficult to
    redissolve.

26. Resuspend in 400 µl s of TE buffer (pH 8.0). Put new, sterile caps
    on the bottles (tightly). Make sure to swirl the buffer around in
    the bottle so that all of the DNA will get resuspended. Leave 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 your tube rack so
    that the rack does not tip over.

27. In the morning, swirl the buffer up and down the sides of the
    bottle, centrifuge the bottles for 1 min (at 14000 rpm), and
    transfer the contents of the bottles into 1.5 ml tubes.

# Reagent/Stock Preparation

70% Ethanol:

Make 500 mls. Use 350 mls of 100% ethanol and 150 mls of DDD water.

Lysis (P2) Buffer:

Make 370 mls. Use 262.5 mls of DDD water, 75 mls of 1N NaOH, and 37.5
mls of SDS (Lauryl Sulfate). Make fresh the day of the large clone
isolation procedure.

P1 Buffer

Add RNAse (100mg/ml) to the P1 buffer solution before starting the large
clone isolation procedure. RNAse once added to the P1 buffer, needs to
be refrigerated at 4ºC.

P3 Buffer

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

Chloramphenicol Preparation:

Add 0.51 grams of chloramphenicol to 15 mls of 100% ethanol for a 34
mg/ml chloramphenicol solution.

Kanamycin Preparation:

Add 0.6 grams of Kanamycin to 30 mls of DDD water to make a 20 mg/ml
kanamycin solution.

TE:

Use pre-mixed Ambiont TE Buffer **OR** mix 1ml of 1M Tris pH 8.0, 200
µls of 0.5M EDTA pH 8.0, and 98.8 mls of DDD water.