Mid-size 8-10kb Library Creation Protocol

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

# Summary

To construct a randomly sheared, non-biased library containing 8-10kb
inserts. *NOTE: When 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/Reagent       | **[                   | **[Stock        |
| s/Equipment]{.underline}** | Vendor]{.underline}** | Number          |
|                            |                       | ]{.underline}** |
+----------------------------+-----------------------+-----------------+
| *                          |                       |                 |
| [Disposables]{.underline}* |                       |                 |
+----------------------------+-----------------------+-----------------+
| Phase Lock Gel             | Eppendorf             | 0032 005.101    |
+----------------------------+-----------------------+-----------------+
| Clear Round Bottom 2-ml    | Eppendorf             | 22 36 335-2     |
| tubes                      |                       |                 |
+----------------------------+-----------------------+-----------------+
| 0.22μM Filter              | Corning               | 430769          |
+----------------------------+-----------------------+-----------------+
|                            |                       |                 |
+----------------------------+-----------------------+-----------------+
| *[Reagents]{.underline}*   |                       |                 |
+----------------------------+-----------------------+-----------------+
| Sodium Hydroxide Pellets   | ACROS Organics/       | 1310-73-2       |
|                            | Fisher Scientific     |                 |
+----------------------------+-----------------------+-----------------+
| Hydrochloric Acid          | Sigma-Aldrich         | H-7020          |
| (concentrated, 12 M)       |                       |                 |
+----------------------------+-----------------------+-----------------+
| 50X TAE Buffer             | Invitrogen            | 24710-030       |
+----------------------------+-----------------------+-----------------+
| 10X Eco Pol Buffer         | New England Bio Labs  | B0210S          |
| (Klenow)                   |                       |                 |
+----------------------------+-----------------------+-----------------+
| 10mM Nucleix Plus dNTP     | MBI Fermentas         | R0192           |
+----------------------------+-----------------------+-----------------+
| T4 DNA Polymerase (100U    | Roche                 | 1004786         |
| 1U/uL)                     |                       |                 |
+----------------------------+-----------------------+-----------------+
| Klenow Large Fragment      | New England Bio Labs  | M0210L          |
+----------------------------+-----------------------+-----------------+
| JGI Mix Marker             | MBI Fermentas         | SM1030          |
+----------------------------+-----------------------+-----------------+
| Marker 2                   | MBI Fermentas         | SM0101          |
+----------------------------+-----------------------+-----------------+
| GenePure LE Agarose        | ISC BioExpress        | E-3120-500      |
| (Generates)                |                       |                 |
+----------------------------+-----------------------+-----------------+
| Ultra Pure Ethidium        | Invitrogen            | 15585011        |
| Bromide (10mg/ml)          |                       |                 |
+----------------------------+-----------------------+-----------------+
| Phenol                     | Sigma                 | P4557           |
+----------------------------+-----------------------+-----------------+
| 5M NaCl                    | Ambion                | 9759            |
+----------------------------+-----------------------+-----------------+
| Pellet Paint               | Novagen               | 69049-3         |
+----------------------------+-----------------------+-----------------+
| 3M Sodium Acetate          | Novagen               | \-              |
+----------------------------+-----------------------+-----------------+
| pMCL200 vector             | JGI                   | \-              |
+----------------------------+-----------------------+-----------------+
| Polyethylene Glycol        | Sigma                 | P-5413          |
+----------------------------+-----------------------+-----------------+
| Glycerol                   | Sigma                 | G-6279          |
+----------------------------+-----------------------+-----------------+
| **[Materials/Reagent       | **[                   | **[Stock        |
| s/Equipment]{.underline}** | Vendor]{.underline}** | Number          |
|                            |                       | ]{.underline}** |
| *[Reagents                 |                       |                 |
| cont.]{.underline}*        |                       |                 |
+----------------------------+-----------------------+-----------------+
| 10X Ligation Buffer        | Roche                 | \-              |
+----------------------------+-----------------------+-----------------+
| 100% Ethanol (200 Proof)   | AAPER Alcohol         | 6810-13289      |
+----------------------------+-----------------------+-----------------+
| TE Buffer pH8.0            | Ambion                | 9849            |
+----------------------------+-----------------------+-----------------+
| T4 DNA Ligase              | Roche                 | 79909           |
+----------------------------+-----------------------+-----------------+
|                            |                       |                 |
+----------------------------+-----------------------+-----------------+
| *[Equipment]{.underline}*  |                       |                 |
+----------------------------+-----------------------+-----------------+
| Hydroshear                 | Gene Machines         | JHSH000000-1    |
+----------------------------+-----------------------+-----------------+
| Hydroshear Large assembly  | Gene Machines         | JHSH204007      |
+----------------------------+-----------------------+-----------------+
| Hydroshear Syringe         | Gene Machines         | JHSH010002      |
+----------------------------+-----------------------+-----------------+
| Juan vacuum centrifuge     | \-                    | \-              |
+----------------------------+-----------------------+-----------------+
| Microcentrifuge (13,500    | \-                    | \-              |
| rpm)                       |                       |                 |
+----------------------------+-----------------------+-----------------+

# Procedure -- Subcloning 

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

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

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

2.  Transfer (be careful to not take from bottom of tube) **5 to 10µg**
    of non-degraded DNA (less DNA may be used if necessary, but results
    may be less constant) to a well-labeled Eppendorf tube; final volume
    **100µl**.

> ***NOTE:** Dilute with TE to bring up to 100μl if necessary.*

3.  Remove reagents from -20^0^C before shearing to ensure rapid
    end-repair after shearing. Confirm that the Klenow buffer
    precipitant is fully dissolved.

4.  Wash the syringe before and after shearing each sample with 0.2M HCl
    (3 times), 0.2M NaOH (2 times), and TE buffer (5 times).

5.  Shear with large assembly:

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a.  Volume = 100µl

b.  \# of cycles = 25 cycles

c.  Speed code = 9 or 10 (may vary by assembly and source of DNA)

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

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

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

1.  To each sheared sample (95-105μl of sheared DNA), add:

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

  10X Eco Pol Buffer (Klenow)                    13 μl

  10mM dNTPs                                     10 μl

  T4 DNA Polymerase (1U/µl)                      6 μl

  Klenow Fragment (5000U/ml)                     6 μl

  Total Volume:                                  **35 μl**
  ---------------------------------------------- ------------------------

\*Make a master mix if needed and spin down.

2.  Cap the well-labeled tube, vortex, and spin down.

3.  Incubate:

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a.  Room temperature for 40 minutes.

b.  70°C for 15 minutes.

c.  4°C (on ice) for 10 minutes.

d.  Freeze at -20°C for 30 minutes before running a gel or store
    overnight at -20°C.

**3. [Size Fractionation/ Agarose Gel Separation]{.underline}**

1.  Make one \~150 ml (almost to arch in between wells) 1.2% TAE agarose
    gel. Use a 12x14cm gel tray and one 8-well comb.

> ***NOTE:** 1L gel contains 12g agarose + \~20µl EtBr and final volume
> to 1L with 1X TAE*.
>
> ![](media/image1.png){width="0.375in"
> height="0.3020833333333333in"}***CAUTION!** Always wear nitrile gloves
> when working with solutions or gels containing ethidium bromide.*

2.  Add **10-20 µl** of 1X loading dye to sample.

3.  Vortex and quick spin down sample.

4.  Load all of the end repaired sample (\~150ul) onto a 1.2% TAE
    agarose gel.

5.  Load with **20μl** of JGI Mix Marker and if there is space, **20μl**
    of Marker 2.

> ***Important!** Keep size markers at least one lane away from sheared
> DNA sample to prevent contamination.*

6.  Run the gel for \~**5 hours at 110-120V** in fresh 1X TAE buffer
    spiked with \~10µl EtBr.

7.  After \~1.5 hours, replace running buffer with new 1X TAE buffer to
    reduce gel temperature (spike new buffer with \~ 10µl EtBr).

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

9.  Visualize gel on long wavelength (360nm) UV box and cut out 6-8kb
    and 8-10kb bands and place them in individual, well-labeled 2-ml
    round bottom Eppendorf tubes.

> ![](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.*

10. Take photo of gel with UV imager after excision of gel slices and
    save photo.

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

1.  Cut off the bottom of a P1000 filter-tip and place in a 1.5-ml tube
    (multiple tips may be needed depending upon size of gel slice).

> ***NOTE:** When cutting tip, be careful it does not crack near filter
> area. The lower you cut below the filter the less likely it will
> split. It just needs to be short enough to fit into an Eppendorf tube
> and put into a centrifuge and close the lid.*

2.  Wash the filter with **50µl** of nuclease-free water.

3.  Spin for **1 minute at maximum speed** (\~12000-14000rpm). Place a
    washed filter-tip into a clean 1.5-ml tube.

4.  Add gel slice to filter-tips making sure not to overload filter
    (make it no higher then even with the top of the filter tip.

5.  Place filter-tip/tube at -20^0^C for 15 minutes.

6.  Spin the filter-tip/tube for **10 minutes at maximum speed**
    (\~12,000-14,000rpm).

7.  Pull out the filter-tip from tube and combine alike eluents in a
    2-ml round bottom Eppendorf tube.

**[Ethanol Precipitation]{.underline}**

1.  Measure sample volume (separate into multiple 2-ml tubes if needed,
    max sample volume is \~560µl).

2.  Add **1/10 volume** of 3M Sodium Acetate, **1.5µl** pellet paint,
    and **2.5 volumes** of room temp 100% ethanol.

3.  Mix gently by inverting several times.

4.  Place on ice for 15 minutes.

5.  Pre-chill microcentrifuge to 4ºC (this takes at least 15 minutes).

6.  Spin tubes at 4°C for **20 minutes at 13,500rpm**.

7.  Dump off the supernatant and discard.

> ***NOTE:** Be careful not to disrupt/lose the faint pink pellet. The
> pellet is pink due to pellet paint.*

8.  Wash the pellet with **500µl** cold 70% ethanol (-20°C).

9.  Spin tube at 4°C for **5 minutes at 13,500rpm**.

10. Carefully pull off all of the ethanol.

> ***Important!** Be careful not to disrupt/lose the faint pink pellet.*

11. Dry the pellet in speed vacuum for 5 minutes using no heat (combine
    pellets before or after drying if needed).

12. Resuspend the pellet in **22µl** T0.1E.

13. Place at 25°C for 5 minutes to fully resuspend pellet (at the half
    way point, vortex lightly or flick the tube to help with
    resuspension).

14. Vortex for 3 seconds and quick spin the tube to collect the sample.

15. If Troubleshooting: QC **2µl** of sample on 1% agarose gel for
    insert size and concentration.

**6. [Ligation]{.underline}**

1.  Make up pMCL200/10X/T4 ligation cocktail:

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

  pMCL200 (\~100ng)                               0.3 µl

  10X Buffer                                      0.8 µl

  Nuclease free H~2~0                             0.7 µl

  T4 DNA ligase                                   0.6 µl

  Total Volume:                                   **2.4 µl**
  ----------------------------------------------- -----------------------

\*\* Make master mix if needed, keep on ice.

2.  Add **4.4µl** (\~100-300ng) of the purified, ethanol precipitated
    DNA to the bottom of a clean, well-labeled 1.5-ml tube.

3.  Aliquot **2.4µl** of ligation cocktail to the bottom of the tube.

4.  Add **1.2µl** of 30% PEG to the bottom of the tube.

5.  Mix well (quick, light vortex or flick a few times) and quick spin
    to collect.

6.  Incubate overnight at 16°C.

**7. [Phenol Extraction]{.underline}**

1.  Prepare phase lock tubes by spinning them down for **2 minutes at
    10,000rpm**.

2.  Bring the sample volume up to **50µl** with T0.1E (42µl).

3.  Add an equal volume (**50µl**) of phenol.

4.  Vortex well for 15-30 seconds.

5.  Add the sample/phenol mixture to the well-labeled phase lock tubes.

6.  Spin tubes for **5 minutes at 10,000rpm**.

7.  Pull off (top) aqueous layer into a clean, well-labeled 1.5-ml tube.

**8. [Ethanol Precipitation]{.underline}**

1.  Add **1/10 volume** 1M NaCl, **1.5µl** pellet paint, and **2.5
    volumes** of room temp 96% ethanol.

2.  Mix well (quick vortex) and quick spin to collect.

3.  Place at -80°C for at least 30 minutes.

> ***NOTE:** While the sample is in the freezer, pre-chill the
> microcentrifuge to 4^0^C ( approx. 15 min).*

4.  Spin at 4°C **for 20 minutes at 13,500rpm**.

5.  Carefully dump off the supernatant and discard.

6.  Wash the pellet with **200µl** of 96% ethanol (cold).

7.  Pull off supernatant. Be careful Not to disrupt/lose the faint pink
    pellet.

8.  Dry pellet in speed vacuum for 5 minutes using no heat.

9.  Resuspend pellet in **20µl** T0.1E.

10. Place at 25°C for 5 minutes to fully resuspend pellet (at the half
    way point, vortex lightly or flick to help with resuspension).

11. Vortex for 3 seconds and quick spin sample to collect sample.

12. Transform immediately or store at -20^0^C.

# 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 Chlor 20 X-gal Plates                         Teknova                    L4415

                                                                              

  *[Reagents]{.underline}*                                                    

  ElectroMAX DH10B Cells (box of 5x100µl ea.)      Invitrogen                 18290015

  SOC Medium                                       Invitrogen                 included

                                                                              

  *[Equipment]{.underline}*                                                   

  Gene Pulser II                                   BioRad                     \-

  Pulse Controller Plus                            BioRad                     \-

  -20^o^C Freezer                                  \-                         \-

  -80^o^C Freezer                                  \-                         \-
  ------------------------------------------------ -------------------------- -----------------------

# Procedure -- Transformation (Electro) 

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

-   Low range: 200

-   High range: ∞

-   Capacitance: 25

-   Voltage: 1.8 kV

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

1.  Place on ice: well-labeled Eppendorf tube and cuvette.

2.  Thaw ElectroMax DH10B competent cells on ice.

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 containing
    ligation (enough cells for 2 transformations (100μl): discard unused
    cells DO NOT refreeze).

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 14-ml
    falcon tube at room temperature (make sure SOC is clear, i.e. no
    growth).

> ***Important!** Transfer the 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 the 1-hour incubation, place the transformation on ice (no
    more than one hour) until ready to plate on agar plates.

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

1.  **Plating with Spreader:**

    a.  When the 60-minute transformation incubation begins, prepare one
        well-labeled LB Chlor 20 X-gal agar plate per library by letting
        them warm to 37^o^C in an incubator to dry agar down and open.

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

    c.  Cap, then mix 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 remaining transformation glycerol stock into cryotube and
    store 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 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 Chlor 20 X-gal
        agar plate per library by letting them warm to 37^0^C in an
        incubator to dry agar down and open (\~ 90 minutes).

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

    c.  Cap, then mix transformation glycerol stock by inverting 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 immediately at --80^0^C for long term storage.

g.  Mix tube by inverting several times (plating tube).

h.  Place 20-30 beads onto bioassay.

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

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

k.  Place bioassay on bench top to dry completely.

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

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

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3.  QC the insert size by PCR or digestion (refer to **Insert Size QC**
    section). If the subcloned DNA has a high GC content (\> 50%), the
    PCR protocol must be adjusted in order for it to work properly
    (refer to **Insert Size QC** section).

# Materials and Reagents -- Insert Size QC

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

  *[Disposables]{.underline}*                                                 

  96-well PCR plate                                \-                         \-

                                                                              

  *[Reagents]{.underline}*                                                    

  Expand Long Template PCR System                  Roche                      1759060

  DNA Polymerase Mix                               Roche                      included

  10X Buffer #3                                    Roche                      included

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

  pUC-R primer (GCAAGGCGATTAAGTTGG)                IDT or Operon or your      R0192
                                                   favorite                   

  TempliPhi 10,000 Rxn kit                         Amersham Biosciences       25-6400-01

  Denaturation Buffer                              Amersham Biosciences       included

  HindIII                                          New England Biolabs        \-

  BamHI                                            New England Biolabs        \-

  10X Buffer 2                                     included                   \-

  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 -- QC of Library Insert Size

NOTE: If the library has a high GC content, adjustments to the standard
PCR protocol must be made or RCA/Digestion may have to be used in order
to determine insert size of the library. If you do not know GC content
and standard PCR does not work, try high GC protocol.

**1.** **[Method 1: Standard 10kb PCR QC]{.underline}**

1.  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~O                  17.0µl                 2040µl

  10X Buffer #3                        2.0 µl                 240 µl

  10mM dNTP (MBI)                      0.4 µl                 48 µl

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

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

  DNA pol mix                          0.2µl                  24 µl

  Total Volume:                        **20.0 µl**            **2400 µl**
  ------------------------------------ ---------------------- ------------------------

2.  Mix and spin down the PCR mix.

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

4.  Seal and spin down the plate.

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

6.  Using pipette tips only, pick the desired number of colonies per
    library (usually 24) into their own well.

7.  Mix tips in cocktail well. Pull out tips carefully 1 or 2 at a time
    to avoid any contamination.

8.  Spin down the plate.

9.  Set up the following colony PCR program on the thermocycler (This is
    an overnight run):

> **94 ^o^C -- 4 min**.
>
> **94 ^o^C -- 15 sec.**
>
> **56 ^o^C -- 30 sec. 10 cycles**
>
> **68 ^o^C -- 10 min.**
>
> **94 ^o^C -- 15 sec.**
>
> **56 ^o^C -- 30 sec. 20 cycles**
>
> **68 ^o^C -- \*10 min.**
>
> **68 ^o^C -- 5 min.**
>
> **4 ^o^C -- Hold**
>
> (\*modify with 5 sec. added on to each cycle for the 68 ^o^C
> elongation step.)

10. Add **10µl** 1X loading dye (must be weak colors so the PCR bands
    are not hidden) to PCR plate.

11. Vortex and spin the plate down to collect.

12. Load **15µl** onto a 1% agarose gel with JGI Mix Marker, and if
    there is space, Marker 2.

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

13. Run for **30 min-1 hour at 120V**.

14. Image gel and check for inserts \~ 8-10kb.

15. Discard the gel in the appropriately labeled waste after the gel is
    imaged.

**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 -- 10 min.**

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

> **58^0^C -- 30 sec. 25 cycles**
>
> **72^0^C -- 10 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 pipette tip) into 96-well PCR plate with **5µl**
    of water in each well (generally, 24 -- 96 colonies per library
    being QC tested).

3.  Seal the plate, vortex gently, and do a 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** (It takes \~ 6 hrs).

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

11. Mix and quick spin down plate.

12. Load **15µl** of sample onto a 1% agarose gel with Wide Range Marker
    ,and if space, Marker 2.

> ***NOTE:** Save the remaining 10µl to use in case there is a problem
> with the QC gel. Can discard after gel is imaged*.

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

14. Image gel and check for inserts 8-10kb.

15. Save image.

#### **3. Method 3: Rolling Circle Amplification/Digestion**

1.  Denaturation:

    a.  Dispense **10µl** of 1X Denaturation Buffer into a 96-well
        sample PCR plate.

    b.  Place in centrifuge for quick spin down.

    c.  Make a visual check to make sure all wells have buffer.

    d.  Using disposable tips, pick 1 colony (white) into each well.

    e.  Mix and carefully pull out tips.

    f.  Place in centrifuge for quick spin down.

    g.  Place plate on thermocycler for 5 minutes at 95^0^C.

    h.  After 5 min denaturation, immediately place plate on ice.

    i.  Cool on ice for at least 5 minutes.

2.  Amplification:

    a.  Remove RCA mix from -80°C freezer before initial denaturation to
        allow it to thaw on ice.

    b.  Gently mix when thawed.

> ***Important!** Do not vortex.*

c.  Dispense **10µl** of RCA mix into each well of the sample PCR plate.

d.  Place in centrifuge for quick spin down.

e.  Make a visual check that all plates have RCA mix.

f.  Place the 96-well PCR plate on the thermocycler with the following
    PCR program:

> **30^0^C -- 20 hrs**
>
> **65^0^C -- 10 min.**
>
> **4^0^C -- hold**

3.  Digestion:

    a.  Aliquot **5µl** of RCA product into a 96-well PCR plate.

    b.  Spin down the plate.

    c.  Make a visual check that all wells have RCA product in them.

    d.  [Make digestion cocktail (Use high concentration
        enzymes):]{.underline}

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

  10X Buffer                          1.0 µl                 120 µl

  Hind III                            1.0 µl                 120 µl

  Bam HI                              1.0 µl                 120 µl

  H~2~O                               2.0 µl                 240 µl

                                      **5.0 µl**             **600 µl**
  ----------------------------------- ---------------------- ------------------------

e.  Dispense **5µl** of digestion cocktail into each well.

f.  Vortex and spin down the plate.

g.  Place the 96-well PCR plate on the thermocycler with the following
    PCR program:

> **37^0^C -- 4 hrs**
>
> **65^0^C -- 15 min.**

**4^0^C -- hold**

h.  Add **10µl** of loading dye to the samples.

i.  Vortex and spin down the samples.

j.  Load **10µl** of digested RCA product onto a 1% agarose gel with
    Wide Range Marker, and if space, Marker 2.

> ***NOTE:** Save the remaining 10µl to use in case there is a problem
> with the QC gel. Can discard after gel is imaged.*

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

l.  Image gel and check for inserts at 8-10kb.

m.  Save image.

# Reagent/Stock Preparation

**[T0.1E:]{.underline}**

40µl 0.5M EDTA

2ml 1M Tris-HCl

197.960ml H~2~0

Adjust if not @ pH 8.0

**[30% PEG:]{.underline}**

3g Polyethylene Glycol

10ml H~2~0

**[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 &                                    
  Development**                                   

  **Instrumentation**                             

  **QC**                                          

  **Purchasing**                                  

  **EH & S**                                      

  **Informatics**                                 

  **Seq Assessment &                              
  Analysis**                                      

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

# Appendix

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

**6/10/04** -- **6/17/04** -- Martin Pollard, Nahal Lalefar, and Bill
Feil performed an audit between 1:00PM (6/10) -- 11:00AM (6/17). The
user was Corey Chinn. Changes incorporated into the protocol on
**8/18/04**.