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ORFV Recombinant Selection

Complete protocol calculator — Infection (MOI), transfection reference, plaque assay serial dilutions (first round & clonal).

🦠 1. Infection Inoculum (MOI) 1 MOI

Cells per well (e.g., 500,000)

Number of wells to infect

MOI (multiplicity of infection)

Virus titer (PFU/mL) e.g., 10⁷·⁹⁴ = 90,909,090

Virus stock required

— mL

neat virus

1:10 dilution volume

— µL

stock + MEM (1:10)

Total inoculum needed

— mL

1 mL per well

MEM to add

— µL

to dilute virus for inoculum

—

🧪 2. Transfection Mix per well

Prepare two tubes, mix, incubate 5 min at RT, then add dropwise to the infected well.

Tube	Reagent	Volume / amount
A	Opti-MEM	125 µL
A	Lipofectamine 3000	7.5 µL
B	Opti-MEM	125 µL
B	Plasmid DNA	2 µg
B	P3000 Reagent	10 µL

⏱ Protocol: Mix Tube B into Tube A, flick to mix, incubate 5 min at RT. Add dropwise to the infected well (total ~270 µL per well).

🔬 3. Plaque Assay 1 — First Serial Dilution 3 plates

Dilute the infection/transfection product as described. Each plate receives 1 mL of the corresponding dilution.

Virus stock for dilution (e.g., 0.5 mL)

mL

Diluent volume – Tube 1 (e.g., 9.5 mL MEM)

mL

Transfer volume & diluent for Tube 2 & 3 (1 mL into 9 mL)

mL into mL

Plate 1 (Tube 1)

—

dilution factor

Plate 2 (Tube 2)

—

dilution factor

Plate 3 (Tube 3)

—

dilution factor

—

🧫 4. Plaque Assay 2 — Clonal 10‑fold Dilutions 10⁻¹, 10⁻², 10⁻³

For each plaque clone, prepare 10‑fold serial dilutions in plain MEM.

Virus stock volume (e.g., 100 µL)

µL

Diluent volume per step (e.g., 900 µL MEM)

µL

Number of dilution steps (e.g., 3)

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