Fosmid (40kb) Library Creation Protocol

> Version Number: 2.1
>
> Production Start Date: 01/28/04
>
> Version 2.1 Date: 08/16/04
>
> Authors: Chris Detter, Saima Shams
>
> Reviewed/Revised by: Eileen Dalin, Jenna Morgan, Hope Tice,
>
> Corey Chin

# Summary

To construct a randomly sheared, non-biased fosmid library containing
\~40kb inserts. *NOTE: When used for whole genomes, only one genome
should be done at a time to prevent mix-ups and cross contamination.*

# Materials & Reagents

+----------------------------+-----------------------+-----------------+
| **[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                      |                       |                 |
+----------------------------+-----------------------+-----------------+
| Falcon 14-ml Polypropylene | Becton Dickinson      | 352059          |
| Tube                       |                       |                 |
+----------------------------+-----------------------+-----------------+
| LB Chloramphenicol 12.5    | Teknova               | L4013           |
| (no X-Gal) plates          |                       |                 |
+----------------------------+-----------------------+-----------------+
| 0.22μM Filter              | Corning               | 430769          |
+----------------------------+-----------------------+-----------------+
|                            |                       |                 |
+----------------------------+-----------------------+-----------------+
| *[Reagents]{.underline}*   |                       |                 |
+----------------------------+-----------------------+-----------------+
| Copy Control Fosmid Kit    | Epicentre             | CCFOS110        |
+----------------------------+-----------------------+-----------------+
| T7 Control DNA (100ng/ul)  | Epicentre             | T7C0204         |
+----------------------------+-----------------------+-----------------+
| Glycerol                   | Sigma                 | G-6279          |
+----------------------------+-----------------------+-----------------+
| MaxPlax Lambda Packaging   | Epicentre             | MP5120          |
| Extract                    |                       |                 |
+----------------------------+-----------------------+-----------------+
| 1M MgCl~2~                 | Ambion                | 9530G           |
+----------------------------+-----------------------+-----------------+
| 1M MgSO~4~                 | Sigma                 | M-3409          |
+----------------------------+-----------------------+-----------------+
| Chloroform                 | Sigma                 | C-2432          |
+----------------------------+-----------------------+-----------------+
| Nuclease-free Water        | Ambion                | 9930            |
+----------------------------+-----------------------+-----------------+
| 0.5M EDTA                  | Ambion                | 9262            |
+----------------------------+-----------------------+-----------------+
| 1M Tris-HCl                | Sigma                 | T-3038          |
+----------------------------+-----------------------+-----------------+
| GenePure Low Melt Agarose  | ISC BioExpress        | E-3126-125      |
+----------------------------+-----------------------+-----------------+
| 10X TBE Buffer             | Invitrogen            | 15581-044       |
+----------------------------+-----------------------+-----------------+
| AgarACE                    | Promega               | M1743           |
+----------------------------+-----------------------+-----------------+
| Phenol                     | Sigma                 | P4557           |
+----------------------------+-----------------------+-----------------+
| 5M NaCl                    | Ambion                | 9759            |
+----------------------------+-----------------------+-----------------+
| Pellet Paint               | Novagen               | 69049-3         |
+----------------------------+-----------------------+-----------------+
| **[Materials/Reagent       | **[                   | **[Stock        |
| s/Equipment]{.underline}** | Vendor]{.underline}** | Number          |
|                            |                       | ]{.underline}** |
| *Reagents cont.*           |                       |                 |
+----------------------------+-----------------------+-----------------+
| 100% Ethanol (200 Proof)   | AAPER Alcohol         | 6810-13289      |
+----------------------------+-----------------------+-----------------+
| SOC Medium                 | Teknova               | 0166-10         |
+----------------------------+-----------------------+-----------------+
| LB Broth                   | Teknova               | L8000           |
+----------------------------+-----------------------+-----------------+
| 0.2M HCl                   | JGI                   | \-              |
+----------------------------+-----------------------+-----------------+
| 0.2M NaOH                  | JGI                   | \-              |
+----------------------------+-----------------------+-----------------+
| TE Buffer                  | Ambion                | 9849            |
+----------------------------+-----------------------+-----------------+
|                            |                       |                 |
+----------------------------+-----------------------+-----------------+
| *[Equipment]{.underline}*  |                       |                 |
+----------------------------+-----------------------+-----------------+
| Hamilton syringe           | \-                    | \-              |
+----------------------------+-----------------------+-----------------+
| Hydroshear                 | Gene Machines         | JHSH000000-1    |
+----------------------------+-----------------------+-----------------+
| Hydroshear Large assembly  | Gene Machines         | JHSH204007      |
+----------------------------+-----------------------+-----------------+
| Hydroshear Syringe         | Gene Machines         | JHSH010002      |
+----------------------------+-----------------------+-----------------+
| Hydroshear Wash Reagents:  |                       |                 |
+----------------------------+-----------------------+-----------------+
| Juan vacuum centrifuge     | \-                    | \-              |
+----------------------------+-----------------------+-----------------+
| Microcentrifuge (13,500    | \-                    | \-              |
| rpm)                       |                       |                 |
+----------------------------+-----------------------+-----------------+
| Chef-DR III System         | BioRad                | \-              |
+----------------------------+-----------------------+-----------------+
| 500-ml Flasks              | \-                    | \-              |
+----------------------------+-----------------------+-----------------+
| Dark Reader                | \-                    | \-              |
| Transilluminator           |                       |                 |
+----------------------------+-----------------------+-----------------+
| -80^o^C freezer            | \-                    | \-              |
+----------------------------+-----------------------+-----------------+
| -20^o^C freezer            | \-                    | \-              |
+----------------------------+-----------------------+-----------------+

# Procedure

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

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

1.  Remove end repair reagents from the --20^0^C freezer to thaw before
    shearing to ensure rapid end repair.

2.  Transfer 20μg of non-degraded DNA to a well-labeled Eppendorf tube
    and bring the final volume to **60µl** with TE, if needed.

> ***NOTE:** To concentrate, dry DNA down to 60µl, if needed.*

3.  Shear the DNA by one of two methods:

    a.  Shear by hand with Hamilton Syringe:

        i.  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). Volume = 60µl.

        ii. \# of passes thru syringe = 4 cycles

        iii. Speed = medium

    b.  Shear with Hydroshear using large assembly:

        i.  Wash the Hydroshear before and after shearing each sample
            with 0.2M HCl (3 times), 0.2M NaOH (2 times), and TE buffer
            (5 times). Volume = 60μl

        ii. \# of cycles = 25

        iii. Speed Code = 17 (may vary by assembly and source of DNA).

4.  Collect the sample in a clean tube and place it on ice immediately.

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

1.  To 50-60µl of sheared DNA, add:

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

  10X Buffer                          **8 µl**

  2.5mM dNTPs                         **8 µl**

  10mM ATP                            **8 µl**

  \*\*Vortex                          

  ER enzyme mix                       **4 µl**

  Total volume                        **88 µl**
  ----------------------------------- -----------------------------------

***NOTE:** Reagents supplied in both the Epicentre fosmid cloning kit
and Epicentre End-it kit.*

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

3.  Incubate at room temperature for 45 minutes.

4.  During the incubation period:

    a.  Prepare a 1% LMP agarose gel (500ml gel contains 5g LMP agarose
        to a final volume of 500ml with 1X TBE).

> ***Important!** DO NOT add Ethidium Bromide.*

b.  Pour a 1% gel at 4^0^C (maximum capacity of tray, without spilling
    over the sides) using a comb containing 5 large wells with 1 small
    well in between each large well (gel needs about 50 min to
    solidify).

c.  After the 45-minute sample incubation, place the sample on a 70°C
    heat block for 10 minutes.

d.  Place the sample on ice until ready to load onto gel.

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

1.  Prepare pulse field gel apparatus:

    a.  Change the 1X TBE Buffer (2.4-2.6L).

    b.  Turn on the pulse field and set the following conditions:

  ---------------------- --------- -----------------------------------------------
  **% Agarose**                    1.0% Low Melting Point Agarose (no EtBr)

  **Buffer**             1X TBE    

  **Temperature**        14^o^C    

  **Voltage**            4.5 V/cm  

  **Pulse**              initial   
                         1.0 --    
                         final 7.0 
                         sec       

  **Run Time**           13 hrs    

  **Angle**              120^0^    
  ---------------------- --------- -----------------------------------------------

2.  When the actual temperature of the pulse field reaches 14^0^C, place
    the gel in the box.

3.  Add **10μl** of loading dye to sample.

4.  Vortex and spin down the sample.

5.  Load the entire sample onto the 1% gel in middle large well.

6.  Load **20μl** of Marker 2 and **20μl** T7 (T7 -- **10μl** T7 +
    **10μl** 1X loading dye) into the other large wells (leave a small
    well in between each sample and marker).

> ***NOTE:** The size markers must be run at least one lane away from
> the DNA sample to prevent cross contamination.*

7.  Run the gel overnight with above parameters.

**4. [Image Gel and Cut out DNA]{.underline}**

> ![](media/image1.png){width="0.375in"
> height="0.3020833333333333in"}***CAUTION!** Always wear nitrile gloves
> when working with solutions or gels containing ethidium bromide.*

1.  Prepare the Ethidium Bromide staining solution (**25µl** of EtBr
    into **500ml** of Milli-Q water).

2.  Add the solution to the staining box.

3.  Using a spatula, remove gel from pulse field platform and place in
    staining solution.

4.  Place the gel on a shaker, using a gentle setting, for 30 minutes to
    stain.

5.  After 30 minutes, drain the staining solution and replace it with
    **500ml** Milli-Q water.

6.  Place the box back on the shaker and destain the gel for 1 hour at a
    gentle setting.

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

8.  ![](media/image1.png){width="0.375in"
    height="0.3020833333333333in"}Use the Dark Transilluminator to view
    bands when cutting.

> ***CAUTION!** Wear cool orange shades.*

9.  Cut out the 40kb band (the band running side by side with the T7
    band, above Marker 2 23kb band) and the band at bottom of well.

10. Place the gel slices in a well-labeled 2-ml round bottom Eppendorf
    tube.

11. Take a photo of the gel with UV imager after excision of gel slices
    and save photo.

**5. [Gel Digestion]{.underline}**

1.  Place the tubes on a 70^0^C heat block to melt the gel completely
    (approximately 5 to 20 minutes).

2.  Remove AgarACE from -20^0^C to thaw.

3.  Place the tubes at 42°C to equilibrate for 3 to 5 minutes.

4.  Divide the melted gel slice into additional tubes with no more than
    **500µl** in each.

5.  Add **6-8µl** AgarACE to each tube based on volume of gel
    \[(1unit/200μl gel) (0.29 units/1μl)\].

6.  Vortex and spin down the tube.

7.  Incubate the tube at 42°C for 20 minutes.

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

> ***NOTE:** Phenol waste should be disposed of in a separate designated
> container.*

1.  Spin for phase lock tubes **2 minutes at 10,000rpm**.

2.  Measure the sample volume.

3.  Add an equal amount of phenol to sample.

4.  Vortex the sample for 15-30 seconds.

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

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

7.  Carefully, pull off (top) aqueous layer into new well-labeled 2-ml
    round bottom tube (avoid touching waxy layer).

**7. [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 1M NaCl, **1.5µl** pellet paint, and **2.5
    volumes** of 96% ethanol.

3.  Vortex well and place tubes at -80°C for at least 30 minutes.

4.  Pre-chill microcentrifuge to 4ºC, this takes at least 15 minutes.

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

6.  Discard the supernatant, keep an eye on the pink pellet.

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

8.  Using a pipet, pull off all the supernatant being careful of "the
    wiley pellet."

9.  Dry the pellet in vacuum for approximately 5 minutes with no heat.
    Combine pellets if needed.

10. Resuspend the pellet with **55µl** T0.1E (volume can be adjusted
    depending upon intensity of band on gel).

11. Vortex and spin down the sample.

12. Place at 25°C for 5 minutes to fully resuspend pellet.

13. Vortex and spin down the sample.

14. If Troubleshooting is needed: QC **2µl** of the sample on 1% agarose
    gel for size and concentration.

15. Store at -20°C until needed.

**8. [Ligation Reaction]{.underline}**

1.  Combine the following in a well-labeled tube (*reagents provided in
    fosmid kit*):

  -------------------------------------------------- ----------------------
                                                     **[2X]{.underline}**

  10X Ligation Buffer                                2.0 µl

  ATP 10mM                                           2.0 µl

  pCC1FOS vector                                     2.0 µl

  insert DNA (40kb)                                  12.0 µl

  DNA Ligase                                         2.0 µl

  Total volume                                       **20 µl**
  -------------------------------------------------- ----------------------

2.  Incubate the tube at room temperature for 2 hours.

3.  Heat-inactivate the sample on the 70^o^C heat block for 10 minutes.

4.  Place the sample on ice for 10 minutes.

5.  Store the tube at -20°C until needed.

**9. [Day 1 Culture (*day before plating*)]{.underline}**

1.  In a 500-ml flask, add **1ml** of 1M MgSO~4~ to **100ml** of LB
    Broth.

2.  Add **5µl** of *E. coli* cells (EPI300 stored at --80^o^C).

3.  Place the culture in shaker at 37°C **overnight (14-18 hrs) at
    200rpm**.

4.  If needed, store the culture at 4°C for up to 5 days.

**10. [Day 2 Culture (*day of plating*)]{.underline}**

1.  In a 500-ml flask, add **1ml** of 1M MgSO~4~ to **100ml** of LB
    Broth.

2.  Add **5ml** of the Day 1 Culture from previous day (make sure Day 1
    is viable).

3.  Place the culture in the shaker at 37°C for **4-6 hours at 200rpm**.

**11. [Packaging:]{.underline}**

1.  In a clean well-labeled tube(s), add **10μl** of ligation.

2.  Thaw 1 tube of packaging extract (1 tube/ 2 reactions 50μl) on ice.

> ***Important!** Do not refreeze unused extract.*

3.  When completely thawed, add **25µl** of packaging extract to
    **10µl** of ligation.

4.  Mix the sample gently with tip and quick spin it down.

5.  Incubate at 30°C for 90 minutes.

6.  Thaw another tube of packaging extract on ice.

7.  When completely thawed, add an additional **25µl** of packaging
    extract.

8.  Mix gently with tip and quick spin down.

9.  Incubate at 30°C for 90 minutes.

10. Add Phage Dilution Buffer to **1000µl** final volume in each tube
    (940μl).

11. Add **25µl** of chloroform to each tube.

12. Gently mix the tube and store it at 4°C until ready to plate (can
    store for up to 30 days).

**12. [Infection / Plating:]{.underline}**

1.  Before the infection, prepare one well-labeled LB--chloramphenicol
    (at 12.5µg/ml no X-gal needed) agar plate per library by letting it
    warm to 37^o^C in incubator to dry open, agar down (1.5 hrs).

2.  Add **100ul** of packaging/phage dilution buffer solution to
    **1000µl** of Day 2 Culture in a 14-ml Falcon tube (can do any 25μl
    : 250μl ratio infection).

3.  Incubate at 37°C for 20 minutes.

4.  After the 30-minute incubation, make a 10% glycerol transformation
    stock (157µl 80% glycerol + full transformation = \~ 1257µl glycerol
    transformation stock).

5.  Cap and mix glycerol stock by inverting it several times.

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

6.  Prepare a tube with **1.5ml** SOC medium (plating tube).

7.  Add **100μl** of the glycerol stock to 1.5ml of SOC (plating tube).

8.  Transfer the remaining glycerol stock into a cryotube and store it
    at -80^0^C for long term storage.

9.  Mix the tube by inverting it several times (plating tube).

10. Place 20-30 beads onto the bioassay.

11. Pipet the entire volume of plating tube onto bioassay with beads
    (\~1600μl).

> ***NOTE:** Pipet the stock over the entire surface of the bioassay.*

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

13. Place the bioassay on the bench top to allow it to dry completely.

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

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

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

# Reagent/Stock Preparation

**[0.2M NaOH]{.underline}**

Mix:

8g NaOH pellets

Bring volume up to 1L with Milli-Q H~2~O.

Filter through a sterile 0.22μM filter

into a sterile 1-L bottle.

**[0.2M HCl]{.underline}**

Mix:

16.7ml of 2M HCl

Bring volume up to 1L with Milli-Q H~2~O.

Filter through a sterile 0.22μM filter

into a sterile 1-L bottle.

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

49.49ml Nuclease-free H~2~0

500µl 1M Tris-HCl (pH

10µl 0.5M EDTA

Adjust if not @ pH 8.0

**[1M NaCl]{.underline}**

10ml 5M NaCl

40ml Nuclease-free H~2~0

**[96% EtOH]{.underline}**

480ml EtOH (200 proof)

20ml Nuclease-free H~2~0

# 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}**

**5/26/04 -- 6/04/04** -- Dan Baker, Christine Lansang, and Nahal
Lalefar performed an audit between 6:00AM -- 11:30 AM. The users were
Saima Shams and Corey Chinn. Changes incorporated into the protocol on
**8/18/04**.