General 3kb Library Creation Protocol

> Version Number: 6.1
>
> Production Start Date: 01/29/04
>
> Version 6.1 Date: 08/18/04
>
> Author: Chris Detter
>
> Reviewed/Revised by: Eileen Dalin, Jamie Jett, Doug Smith

# Summary

This protocol is for the high-throughput production of subcloned BAC DNA
libraries. The starting material is generally purified BAC DNA with an
average length of 120kb and the finished products are arrayed microtiter
plates of subclones with an average length of 3kb that are ligated into
pUC18 vector and transformed into ElectroMAX DH10B competent cells.

This protocol may also be used, after minor modification, to subclone
microbial genomes, as well as other larger whole genomes. *NOTE: If used
for whole genomes, only one genome should be done at a time to prevent
mix-ups and cross contamination.*

# Materials and Reagents -- Subcloning

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

  *[Disposables]{.underline}*                                                 

  QIAquick Gel Extraction Kit (50) **OR**          QIAGEN                     28704

  QIAquick Gel Extraction Kit (250)                QIAGEN                     28706

  0.22μM Filter                                    Corning                    430769

                                                                              

  *[Reagents]{.underline}*                                                    

  Sodium Hydroxide Pellets                         ACROS Organics/ Fisher     1310-73-2
                                                   Scientific                 

  Hydrochloric Acid (concentrated, 12 M)           Sigma-Aldrich              H-7020

  GenePure LE Agarose (Generates)                  ISC BioExpress             E-3120-500

  50X TAE Buffer                                   Invitrogen                 24710-030

  T4 DNA Polymerase 1U/µl                          Roche                      1004786

  DNA Polymerase I (Klenow) 5000U/ml               NEB                        M0210L

  10X Klenow Buffer (EcoPol)                       NEB                        B0210S

  Fast-Link DNA Ligation Kit                       Epicentre                  LK6201H

  10mM dNTP mix                                    MBI Fermentas              R0192

  pUC18 SmaI / SAP                                 Roche + JGI                0885819 + JGI cut

  Glycerol                                         Sigma                      G-6279

  Marker 2                                         MBI Fermentas              SM0101

  Marker 4 (altered at JGI for 3-4kb bands)        MBI Fermentas              SM0291

  TE Buffer pH8.0                                  Ambion                     9849

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

  *[Equipment]{.underline}*                                                   

  Agarose gel box and two 13-teeth combs           Owl                        D-14

  Hydroshear                                       Genomic Solutions          JHSH000000-1

  Standard Hydroshear Shearing Assembly            Genomic Solutions          JHSH204004

  Hydroshear Syringe                               Genomic Solutions          JHSH010002
  ------------------------------------------------ -------------------------- -----------------------

# Procedure -- Subcloning 

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

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

1.  Remove samples (1-12) from the --80^0^C freezer, and let thaw.

2.  Vortex and spin isolated DNA for **5 minutes at 12,000rpm**.

3.  Pipette **100µl** into a new micro centrifuge tube (be careful to
    not take from bottom of tube).

4.  Shear **100µl** of \~30ng/µl isolated BAC DNA (\~3µg total) with the
    Hydroshear.

5.  Open the Hydroshear program and set the shearing parameters as
    follows:

    a.  DNA volume = 100µl

    b.  \# of Cycles = 25

    c.  Speed Code = 12 or 13 (qc each Hydroshear for size)

    d.  Wash Cycles: 3x with 0.2M HCl, 2x with 0.2M NaOH, 5x with TE.

6.  After shearing, collect the sample(s) into microcentrifuge tube(s).
    Place each tube on ice immediately after shearing.

> ***NOTE:** The volume recovered after shearing should be approximately
> 95-105µl.*

7.  Remove reagents from the --20^o^C freezer when 3 or 4 samples are
    left to shear, to ensure rapid end-repair after shearing. Confirm
    that the Klenow buffer precipitant is fully dissolved.

**2. [Blunt End Repair]{.underline}**

1.  End repair **95-105µl** of each sheared sample in a microcentrifuge
    tube. To each sample, add:

  ---------------------------------- ---------------------- -----------------------
                                     **[1X]{.underline}**   **[13X]{.underline}**

  10X Klenow Buffer (EcoPol)         13 µl                  169 µl

  10mM dNTPs                         10 µl                  130 µl

  T4 DNA Polymerase (1U/µl)          6 µl                   78 µl

  Klenow Fragment (5000U/ml)         6 µl                   78 µl

  Total Volume                       **35 µl**              **455 µl**
  ---------------------------------- ---------------------- -----------------------

> \*A well-mixed cocktail of the above can be prepared (***store on
> ice***) and aliquoted if needed.
>
> \*\*\*Add 35µl of the end-repair master mix to each tube containing
> 95-105µl of sheared DNA.

2.  Mix/vortex the tube for \~3 seconds.

3.  Quick spin the tube.

4.  Allow the reaction to proceed at room temperature (on bench top) for
    40 minutes.

5.  After 40 minutes, place the tube on a hot block at 70^o^C for 15
    minutes, then let it cool at 4^o^C (on ice) for 5 minutes.

6.  The tube must be frozen at -20^o^C (freeze) for at least 30 minutes
    or can be stored overnight at -20^o^C.

**3. [Agarose Gel Separation]{.underline}**

1.  Make one \~350ml (almost to arch in between wells) 1% TAE agarose
    gel. Use a 23cm x 14cm gel tray and two 13-teeth combs for every 12
    DNA samples.

> ![](media/image1.png){width="0.375in"
> height="0.3020833333333333in"}***NOTE:** A 1L gel contains 10g
> agarose + 20µl EtBr. Bring final volume to 1L with 1X TAE.*
>
> ***CAUTION!** Always wear nitrile gloves when working with solutions
> or gels containing ethidium bromide.*

2.  Thaw the end-repaired samples and add **10-20µl** of a 1X loading
    dye to each tube.

3.  Vortex and quick spin to collect.

4.  Change the buffer in the gel box before use.

5.  Load all of the end-repaired product (\~ 150μl) onto the 1.0%
    agarose gel (if the wells are a little shallow and the sample begins
    to overflow, stop loading sample).

> ***NOTE:** Load a sample every other well, total of six samples per
> comb.*

6.  Load **10µl** of the lambda HindIII size marker (Marker 2 only) into
    the left, middle, and right small wells, load **20µl** into large
    adjacent wells.

> ***NOTE 1:** For whole genomes, also use JGI altered Marker 4, which
> shows (2.7kb, 3.3kb, and 4.2kb bands). This will allow for more
> precise cutting out of DNA.*
>
> ***NOTE 2:** For whole genomes, keep size markers at least one lane
> away from sheared DNA sample.*

7.  Run the gel for \~**60 minutes at 110-120V**.

> ***NOTE:** It is very important that you get visible separation
> between the 2.0kb and 2.3kb size marker bands. After this happens, you
> can image and easily cut out the desired \~3-4kb band for each
> sample.*

8.  Image the gel (image for no longer than 5 seconds to minimize UV
    exposure) and save photo.

9.  For BAC subcloning, cut the gel into 2 or 4 parts to minimize UV
    exposure to samples other than the one being excised. Cut out each
    gel fragment between the 2.3kb and 4.3kb size marker using a clean
    razor blade (each slice weighs \~180mg). The thinner the band the
    more consistent the insert size will be. Put each slice into its own
    well-labeled 1.5-ml microfuge tube.

> ![](media/image1.png){width="0.375in"
> height="0.3020833333333333in"}***CAUTION!** Minimize UV exposure to
> yourself (use long sleeves and face shield) and to the DNA samples.
> Use the blue laminate to shield samples not being extracted and turn
> off UV after cutting out each band.*
>
> ***NOTE:** For whole genomes cut a thin slice at the 3.3kb Marker 4
> band.*

10. Image the gel after 3-4kb band extraction and save photo.

**4. [Gel Purification Filter]{.underline}**

1.  Add **600µl** of Buffer QG to each tube containing a gel slice,
    close top and mix by hand a few times by shaking back and forth.

> ***NOTE:** Make sure gel and QG buffer are at the bottom of tube when
> done.*

2.  Incubate each tube in the heating block at 40^o^C shaking at 800rpm
    until the gel is completel**y** melted (**\~**10-15 minutes) (half
    way through shake tube back and forth with hand to mix again to help
    dissolve the gel quicker).

> ***NOTE:** Make sure gel is dissolved, must be yellow in color to have
> proper pH. If the color is pink, adjust the pH with **10µl** of 3M
> Sodium Acetate, pH5. This rarely occurs.*

3.  Following the Qiagen protocol steps: add the **\~750µl** sample to
    the Qiagen spin columns. Spin for **1 minute at 13,000rpm**. Dump
    waste from the bottom of the waste tube.

> ***NOTE:** If the sample is more than 750µl, spin, dump, and repeat
> with remaining sample.*

4.  Add **750µl** of wash buffer PE to each spin column. Let sit for 5
    minutes. Spin for **1 minute at 13,000rpm**. Dump the waste from the
    bottom of the waste tube.

5.  Respin for **1 minute at 13,000rpm** to dry spin column.

6.  To elute DNA, put spin columns in new, labeled 1.5-ml tubes and add
    **50µl** of warm (\~40-50^o^C) Buffer EB. Reduce the EB volume used
    to 30µl if the bands you cut out are faint.

7.  Let sit 1 minute then spin for **1 minute at 13,000rpm**.

8.  Vortex and spin down sample.

9.  If Troubleshooting is needed: QC **5µl** of each sample on gel with
    ladder and concentration standard to confirm size and quantity.

**5. [Vector Ligation]{.underline}**

1.  In a clean, well-labeled PCR plate, add **1µl** of pUC18 SmaI/SAP
    vector (\~100ng) to each well.

> ***NOTE:** The pUC18 vector is from Roche and was cut (with SmaI) and
> prepared (dephosphorylated with SAP, and gel sized selected and
> purified) at the JGI.*

2.  Transfer **10µl** of the \~3kb size selected gel purified BAC DNA
    sample (\~200ng) into a designated well of a 96-well PCR plate.

3.  In a 1.5-ml centrifuge tube on ice, prepare a cocktail mix of:

  ------------------------------------------- ---------------------- -----------------------
                                              **[1X]{.underline}**   **[15X]{.underline}**

  10X Ligation Buffer (Fast-Link) per sample  1.50 µl                22.50 µl

  10mM ATP (Fast-Link) per sample             0.75 µl                11.25 µl

  Fast-Link DNA Ligase per sample             2.00 µl                30.00 µl

  Total Volume                                **4.25 μl**            **63.75 μl**
  ------------------------------------------- ---------------------- -----------------------

\*\* Vortex well and quick spin the tube before dispensing.

4.  Dispense **4.25µl** of cocktail mix to each sample in the PCR plate
    (on the side wall of each well). Seal, spin down plate and mix
    gently. Quick spin down plate.

5.  On a thermocycler, set up the following ligation protocol times:
    *24.5^o^C -- 90 min, 70^o^C -- 15 minutes, and hold at 4^o^C*. The
    reaction can stay overnight at 4^o^C if needed, but should be stored
    at -20^o^C for long-term storage.

# Materials and Reagents -- Transformation

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

  *[Disposables]{.underline}*                                                 

  Gene Pulse Cuvette 0.1 cm electrode gap          BioRad                     165-2089

  Falcon 14 ml Polypropylene Tube                  Becton Dickinson           352059

  Cryogenic Vial                                   Corning                    430289

  LB Amp 100 X-gal Plates                          Teknova                    L4982

                                                                              

  *[Reagents]{.underline}*                                                    

  ElectroMAX DH10B Cells (box of 5 x 100 µl ea.)   Invitrogen                 18290015

  SOC Medium                                       Teknova                    S1640

                                                                              

  *[Equipment]{.underline}*                                                   

  Gene Pulser II                                   BioRad                     \-

  Pulse Controller Plus                            BioRad                     \-
  ------------------------------------------------ -------------------------- -----------------------

# Procedure -- Transformation (Electro) 

**1. [Equipment Settings (BioRad Pulse Controller)]{.underline}**

-   Low range: 200

-   High range: ∞ (not used)

-   Capacitance: 25

-   Voltage: 1.8 kV

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

1.  Place on ice: one well-labeled Eppendorf tube and cuvette for each
    sample being transformed.

2.  Thaw ElectroMax DH10B competent cells on ice (each tube contains
    100µl, enough for 2 reactions). Discard unused cells-DO NOT
    refreeze!

3.  To the appropriately well-labeled, cold Eppendorf tube; add **1µl**
    of ligation product.

4.  Once thawed, mix competent cells by swirling with pipette tip a few
    times.

5.  Add **50µl** eDH10B competent cells to the Eppendorf tube.

6.  Mix by swirling the ligation and competent cells together with
    pipette tip a few times.

7.  Transfer solution to the bottom groove of COLD cuvette and tap on
    tabletop a few times to settle solution to the bottom (must see even
    levels of cells on each side of the cuvette without bubbles).

8.  Electroporate at 1.8kV.

9.  Transfer cell solution immediately to **950µl** of SOC in a 14ml
    falcon tube at room temperature (make sure SOC is clear, i.e. no
    growth).

> ***Important!** Transfer electroporation solution within 10 seconds.*

10. Rinse cuvette with **50µl** of the same SOC mixture to which you
    just added the cells.

11. Incubate within rotating wheel at 37 ^o^C for 1 hour.

12. After incubation, place on ice (no more than one hour) until ready
    to plate on agar plates.

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3.  **[Plating]{.underline}**

    1.  **Plating with a Spreader:**

        a.  When the 60-minute transformation incubation begins, prepare
            one well-labeled LB/Amp/IPTG/X-gal agar plate per library by
            letting the plate warm to 37^o^C in an incubator to dry agar
            down and open.

        b.  After the 60-minute transformation incubation, make a 10%
            glycerol transformation stock (143µl 80% glycerol + full
            transformation = \~1143µl glycerol transformation stock).

        c.  Cap and mix the stock by inverting several times.

**NOTE:** You can store the tube at -80 ^o^C if not plating within 10
minutes.

d.  First pipette \~**500µl** SOC onto the center of the plate, then add
    **5µl** of transformation to the center with the SOC.

e.  Spread in a small circular motion at first to help mix, and then
    spread evenly across the entire plate.

f.  Transfer the remaining transformation glycerol stock into a cryotube
    and store it immediately at -80 ^o^C for long-term storage.

g.  Incubate the plates agar down in 37^o^C incubator for 16\~18 hrs.

h.  Count the colonies and determine the complexity of ligation reaction
    (total \# of colonies in ligation).

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2.  **Plating with Beads:**

    a.  Before transforming, prepare one well-labeled LB/Amp/IPTG/X-gal
        agar plate per library by letting the plate warm to 37^0^C in an
        incubator to dry agar down and open (\~90 min).

    b.  After 60-minute transformation incubation, make a 10% glycerol
        transformation stock (143µl 80% glycerol + full transformation =
        \~1143µl glycerol transformation stock).

    c.  Cap and mix the transformation glycerol stock by inverting it
        several times.

**NOTE:** You can store the tube at -80 ^o^C if not plating within 10
minutes.

d.  Prepare a tube with **1.5ml** of SOC (plating tube).

e.  Add **5μl** of the transformation glycerol stock to 1.5ml of SOC
    (plating tube).

f.  Transfer remaining transformation glycerol stock into cryotube and
    store it immediately at --80^0^C for long-term storage.

g.  Mix the plating tube by inverting several times.

h.  Place 20-30 beads onto bioassay.

i.  Pipet the entire volume of plating tube onto bioassay with beads
    (\~1505μl). Pipet the stock all over plate.

j.  Gently shake the beads around on bioassay until the stock has been
    evenly spread.

k.  Place the bioassay on the bench top to dry completely.

l.  When the bioassay is dry, pour the beads off of the bioassay into
    autoclave waste.

m.  Incubate the bioassays agar down in 37^0^C incubator for 16-18 hrs.

# Materials and Reagents -- PCR QC

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

  *[Disposables]{.underline}*                                                 

  96-well PCR plate                                USA Scientific             1402-9708

                                                                              

  *[Reagents]{.underline}*                                                    

  Taq DNA Polymerase (10KU)                        Amersham Biosciences       27-0799-63

  10X PCR Buffer                                   Amersham Biosciences       \-

  10mM dNTP mix                                    MBI Fermentas              R0192

  pUC-F primer (CTTTACACTTTATGCTTCC)               IDT or Operon or your      \-
                                                   favorite                   

  pUC-R primer (GCAAGGCGATTAAGTTGG)                IDT or Operon or your      \-
                                                   favorite                   

  FailSafe PCR System (Enzyme mix for high GC)     Epicentre                  FS99250

  (choose 2x Buffer K)                             Epicentre                  FSP995K

                                                                              

  *[Equipment]{.underline}*                                                   

  GeneAmp PCR System 9700                          Perkin Elmer (Applied      \-
                                                   Biosystems)                
  ------------------------------------------------ -------------------------- -----------------------

# Procedure -- PCR for QC of Library Insert Size

**1.** **[Method 1: Standard 3kb PCR QC]{.underline}**

1.  Make up the following PCR master mix for each sample (x120 for a
    96-well plate): Keep plate on ice.

  ------------------------------------- ---------------------- ------------------------
                                        **[1X]{.underline}**   **[120X]{.underline}**

  Nuclease free H~2~O                   16.85 µl               2022.00 µl

  10X PCR Buffer                        2.00 µl                240.00 µl

  10mM dNTP (MBI)                       0.40 µl                48.00 µl

  pUC-F primer (10 pmol/µl)             0.28 µl                33.60 µl

  pUC-R primer (10 pmol/µl)             0.28 µl                33.60 µl

  Taq Polymerase (Amersham)             0.28 µl                33.60 µl

  Total Volume:                         **20.09 µl**           **2410.8 µl**
  ------------------------------------- ---------------------- ------------------------

2.  Dispense **20µl** of the PCR mix into each well needed of the PCR
    plate. Keep plate on ice.

3.  Spin down the plate.

4.  Make a visual check to make sure all of the wells have mix in them.

5.  Using pipette tips only, pick the desired number of colonies per
    library (usually 16 colonies per library) into their own well. Mix
    tips in the cocktail thoroughly.

> ***Important!** Be careful of your well location and of
> contamination.*

6.  Quick spin the plate.

7.  Set up a the following colony PCR program on the thermocycler (This
    is a 3hr 15min protocol):

> **94 ^o^C -- 4 min.**
>
> **94 ^o^C -- 30 sec.**
>
> **55 ^o^C -- 30 sec. 35 cycles**
>
> **68 ^o^C -- 2 min.\***
>
> **4 ^o^C -- Hold**
>
> (\*modify with 5 sec. added on to each cycle for the 68 ^o^C
> elongation step.)

8.  Add **10µl** loading dye (must be faint so that the PCR bands are
    not hidden) to PCR plate.

9.  Vortex and spin down plate to collect.

10. Load **15µl** of sample onto gel and save the remaining sample for
    later use if gel fails. Leave a well empty between libraries for
    loading **5µl** of Marker 2.

11. Run the gel for \~ **30 minutes at 120V**.

12. Image the gel and check for inserts \~3kb.

13. Discard the gel in the appropriately labeled waste.

**2. [Method 2: High GC PCR QC]{.underline}**

1.  Set up a PE 9700 with the following colony PCR program (overnight
    run):

> **98^0^C - 3 min.**

**98^0^C - 40 sec.**

> **58^0^C -- 30 sec. 10 cycles**
>
> **72^0^C -- 3 min.**

**98^0^C -- 40 sec.**

> **58^0^C -- 30 sec. 25 cycles**
>
> **72^0^C -- 3 min.\***
>
> **4^0^C -- Hold**

(\*modify with 5 sec. added on to each cycle for the 72^0^C elongation
step)

2.  Pick colonies (with a pipette tip) into 96-well PCR plate with
    **5µl** of water in each well.

> ***NOTE:** We generally do 24 -- 96 colonies per library being QC
> tested.*

3.  Seal plate, vortex gently, and quick spin.

4.  Incubate at 95^0^C for 5 minutes on the thermocycler, remove and
    cool on ice for 5 minutes.

5.  Spin plate for **5 minutes at 4,000rpm**.

6.  Make up the following PCR master mix for each sample (x120 for a
    96-well plate): Keep on ice.

  ------------------------------------ ---------------------- ------------------------
                                       **[1X]{.underline}**   **[120X]{.underline}**

  Nuclease-free H~2~0                  6.6 µl                 792 µl

  2x pre-mix Buffer K                  10.0 µl                1200 µl

  pUC-F primer (10pmol/µl)             0.2 µl                 24 µl

  pUC-R primer (10pmol/µl)             0.2 µl                 24 µl

  FailSafe Enzyme mix                  1.0 µl                 120 µl

  Total Volume:                        **18.0 µl**            **2260 µl**
  ------------------------------------ ---------------------- ------------------------

7.  Aliquot **18µl** of PCR cocktail (from step **2.6**) into the
    desired number of wells of a new 96-well PCR plate (Keep on ice).

8.  Using colony samples from step **2.2**, transfer **2µl** from top of
    well to the PCR plate containing the PCR cocktail from step **2.7**.

9.  Seal plate and cycle on the thermocycler using conditions in Step
    **2.1** (This takes \~ 6 hrs).

10. Add **10µl** of loading dye to PCR plate.

11. Mix and quick spin down plate.

12. Load **15µl** of sample onto a 1% agarose gel with Marker 2.

> ***NOTE:** Save the remaining to use in case there is a problem with
> the QC gel. You can discard the remaining sample after the gel is
> imaged*.

13. Run for \~**30 minutes at 120V**.

14. Image gel and check for inserts \~3kb.

15. Save image.

# Reagent/Stock Preparation

**[80% Glycerol Stock Solution]{.underline}**

40ul 100% Glycerol (pipette slowly)

10ul Nuclease-free H~2~0

Autoclave

**[6X Loading Dye]{.underline}**

75ml 100% glycerol

125ml Nuclease-free H~2~0

0.05g Bromophenol Blue

0.05g Xylene Cyanole FF

**[1X Loading Dye]{.underline}**

400ul Nuclease-free H~2~0

100ul 100% glycerol

100ul 6X loading dye

**[1X TAE Buffer]{.underline}**

To make 20 liters:

400ml 50X TAE Buffer

19.6L Milli-Q H~2~O

# SOP Approval

  ----------------------- ----------------------- -----------------------
  **DEPARTMENT**          **APPROVED BY**         **DATE**

  **Lab Supervisor**                              

  **Research and                                  
  Development**                                   

  **Instrumentation**                             

  **QC**                                          

  **Purchasing**                                  

  **EH and S**                                    

  **Informatics**                                 

  **Seq Assessment and                            
  Analysis**                                      

  **Dept Head of Prod                             
  Seq**                                           
  ----------------------- ----------------------- -----------------------

# Appendix

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

**6/07/04 -- 6/15/04** -- Tijana Glavina and Jamie Jett performed an
audit between 9:30AM (6/07) -- 10:30AM (6/15). The user was Doug Smith.
Changes incorporated into the protocol on **8/18/04**.