Protocol: Spi^- Assay of gpt delta Transgenic Mouse 
(Ver.2.1en/2002.05.24)

Preparation of Plating Bacteria

1. One day before the mutation assay, inoculate LB broth with E. coli XL-1Blue MRA and XL-1Blue MRA (P2), and incubate them overnight at 37^oC with shaking.

2. Next morning, inoculate LB + 0.2% (v/v) maltose with 1/100 vol. of the overnight cultures.

3. Incubate at 37^oC with shaking until OD₆₀₀ reaches 1.0.

4. Centrifuge and resuspend the pellets with equal vol. of LB + 10 mM MgSO₄.

5. Store the suspended cells at 4^oC until use. (the concentrated suspensions of XL-1 Blue MRA and XL-1Blue MRA(P2) are used for titration, Spi^- mutant selection and confirmation of mutant.)

In vitro Packaging Reactions

< Transpack Packaging Extract (STRATAGENE, La Jolla, CA) >

Please see the original protocol provided from STRATAGENE. Adjust the final volume to 300 microL by SM buffer.

<HOMEMADE Packaging Extract>
1. Mix DNA samples by pipetting with wide-bore pipette (we use the product of QALITY SCIENTIFIC PLASTICS, QSP #: 118-96RN).
2. Thaw SE at room temperature and then thaw FTL with finger tips. Thaw each one of SE and FTL for every two packaging reactions.
3. Immediately aliquot half (15 microL) of FTL into a fresh 1.5 mL eppendorf tube.
4. Add genomic DNA sample (5-7.5 microL) to the 15 microL of FTL.
5. Mix by pipetting with the wide-bore pipettes about 20 times, avoiding introduction of air bubbles.
6. Add SE (30 microL) with the wide-bore pipettes. Mix well.
7. Incubate the tubes at 37^oC for 90 min. The remaining SE (30 microL) and FTL (15 microL) are frozen again on dry ice.
8. Thaw re-frozen FTL (15 microL), and add it to the reaction and mix several times.
9. Thaw re-frozen SE (30 microL), and add it and mix several times.
10. Incubate at 37^oC for an additional 90 min.
11. Add SM buffer and adjust the final volume to 300 microL. Mix by gentle vortexing.
12. Keep them at 4^oC or use immediately.

Titration of the Packaged Phage with Strain XL-1Blue MRA 

1. Take the packaged phage suspension (5 microL) and mix with LB broth (495 microL) to make a 100-fold diluted suspension. 

2. Add 10 microL of the diluted suspension to 200 microL of the  XL-1Blue MRA suspension. We use sterile glass tubes (1.2 cm x 7.5 cm) for this assay.

3. Incubate for 20 min at room temperature.

4. Add 2.5 mL of molten l-trypticase soft agar.

5. Pour on l-trypticase agar plates (f 9 cm x 2 plates). 

6. Incubate overnight at 37^oC.

7. Count the number of plaques and calculate the number of p.f.u. (plaque forming unit) per packaging reaction.

[ Titer = Number of plaques (average of 2 plates) × Dilution Factor ]
Dilution Factor = 3000

The Spi^- assay of the packaged phage using XL-1Blue MRA(P2)

1. Add 150 microL of the remaining packaged phage suspension to 200 microL of the  XL-1Blue MRA(P2) suspension. We use 2 sterile glass tubes (1.2 cm x 7.5 cm) for this assay.

2. Mix by gentle vortexing.

3. Incubate for 20 min at room temperature.

4. Add 2.5 mL of molten l-trypticase soft agar to each tube.

5. Pour on l-trypticase agar plates (9 cm x 2 plates).

6. Immediately, remove the bubbles on the agar layer by exposing to gas fire for 1-2 seconds.

7. Incubate overnight at 37^oC. 

8. Count the number of clear plaques on the 2 plates (Spi^- candidates).

Confirmation of the Spi^- phenotype 

1. One day before the spot test, inoculate LB broth with E. coli WL95(P2), and incubate them overnight at 37^oC with shaking. Centrifuge the overnight culture and resuspend the pellets with equal vol. of LB+10 mM MgSO₄.

2. Punch out the clear plaques (Spi^- candidates) with sterilized glass capillary.

3. Suspend the agar plug in 60 microL of SM buffer.

4. Spot 15 microL of the suspension each on three l-trypticase plates where each of XL-1Blue MRA, XL-1Blue MRA(P2) and WL95(P2) strains is spread with l-trypticase soft agar.

5. Incubate overnight at 37^oC.

6. Count the number of real Spi^- mutants. If the Spi^- candidates are real Red^-/Gam^- mutants, they should make clear spots on all of three strains.
(On the XL-1Blue MRA(P2) plate, minor types of mutants, which are Gam^- mutants without deletion, may also make plaque. By using another P2 lysogen, WL95(P2), Red^-/Gam^- mutants can be selected.)

7. Calculate the mutant frequency by dividing the total number of real Spi^- plaques (Red^-/Gam^- mutants) by the number of total plaques recovered.

Preparation of the Lysate of the Spi^- Phage

1. Inoculate LB broth with E. coli LE392 and incubate overnight at 37^oC with shaking.

2. Spin down the overnight culture and suspend the pellet in equal vol. of LB+10 mM MgSO₄.

3. Punch out the clear spot (the real Spi^-) on agar plates with capillary and add the agar plug to 50 microL of the E. coli suspension.

4. Stand for 5 min at room temperature.

5. Add 2.5 mL LB broth + 10 mM MgSO₄.

6. Incubate at 37^oC with vigorous shaking until lysis occurs (about 7-8 hours).

7. Add 100 microL  chloroform.

8. Centrifuge at 15, 000 rpm for 10 min.

9. Take the supernatant and keep it at 4^oC.

Check the Titer of the Spi^- Lysate

1. The lysate is diluted 10⁶ fold with LB broth.

2. The diluted lysate (10 microL) is mixed with 200 microL of the 2-fold concentrated cell suspension of XL-1Blue MRA or E. coli C  resuspended overnight culture with +10 mM MgSO₄.

3. Add 2.5 mL of molten l-trypticase soft agar.

4. Pour on l-trypticase plates.

5. Incubate overnight at 37^oC.

6. Count the number of plaques.

7. Calculate the p.f.u. per mL of the lysate.

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> l-trypticase agar plate (1 L)
BBL trypticase peptone (Becton Dickinson) 10 g
NaCl 5 g
Agar (Difco) 10 g
H₂O 1 L
Sterilize by autoclave. Allow the solution to cool to 60^oC, and add 1/100 vol. of 1 M MgSO₄ to final conc. 10 mM.

## Agar conc. is 1%. The plates should not be dried. Fresh and wet plates are preferable. The volumes of agar plate are about 25 ml (Eiken Sterile Auto Schale, 9 cm diameter). 

> l-trypticase top agar (100 mL)
BBL trypticase peptone (Becton Dickinson) 1 g
NaCl 0.5 g
Agar (Difco) 0.6 g
H₂O 100 mL
Sterilize by autoclave. Before using, melt again by short-time autoclaving (120^oC, 5min) and shake well. Keep it in water bath at 50^oC before and during use. Add 1/100 vol. of 1 M MgSO₄ to final conc. 10 mM.

## Addition of MgSO₄ in bottom and top agar improves the plaque formation.
## It will be difficult to identify Spi^- plaques after overnight incubation if there are small bubbles on the top agar layer. Thus, small bubbles on the top agar layer should be completely removed out as soon as pouring the molten soft agar on the plates. It can be done by exposing the top agar layer to gas fire 2-3 seconds. Exposure of the agar plates containing phage and bacteria to gas fire for 2-3 seconds does not affect the plaque formation.