VSV Rescue Using VVT7

VSV Rescue Using VVT7

VSV Rescue using VVT7

Cut cells day before: ____ 6-well plates (1 per mutant) BHK/A549 1:_____. Use media without Pen/Strep.

Well #1= mock transfected, wells #2-6 plasmids

Day of experiment:

  1. Aspirate media, wash with PBS (1.5ml/well)
  2. Infect with VVT7 at MOI=5 in DMEM w/o serum (0.5ml/well; no P/S)
  • VVT7 batch ____, titer =______
  • Amount/well:______ul → Use _____ul VVT7 in ______ml SFM +0.1ml
  • Distribute 0.5ml VVT7/SFM mix to each well. Incubate at 37C for 1hr with rocking every 15min.
  1. During infection, set up transfection mixes in polystyrene tubes
  • Set up tubes with Lipofectamine in Opti-MEM (no Pen/Strep) as indicated in the last column below at 6.6x per plate. (Standard is 3ul Lipo/ug DNA, total vol. = 1ml/well) Pipette up and down gently to mix. Incubate at RT for 15 min.
  • While Lipo/Opti is incubating, add DNA to Opti-MEM (no P/S) for a final volume of 1ml/well. Make a master mix of 5.5x rxn/mutant. Place 1ml of Opti-MEM in separate tube for mock transfected well.
  • Add equal volume Lipo/Opti mix to DNA/Opti mix and pipette gently up and down to mix. Incubate at RT for 30-45min.
  1. Aspirate VVT7 from plates and wash with 1.5ml Opti-MEM per well. Add transfection mixes to appropriate wells (2ml per well).
  2. Incubate for 4hrs at 36C
  3. Aspirate media and add 2ml MEM-BHK with 5% FBS to each well. Continue incubation at 36C. Watch for CPE (usually not visable before 1st pass)

Well # / DNAs (pBS) / DNA/Opti Mix / Lipo/Opti Mix
5µg Pwt
µg / µl / 3 µg Nwt
µg / µl / 1 µg Lwt
µg / µl / 5 µg FL
µg / µl / DNA master mix (1ml/well) / Lipo
42µl/well / Opti
958µl/well
1
2 / P:
3 / N:
4 / L:
5 / FL:
6 / Opti
7
8 / P:
9 / N:
10 / L:
11 / FL:
12 / Opti
13
14 / P:
15 / N:
16 / L:
17 / FL:
18 / Opti

Pass 1

2 days after transfection:

Cut ____ 6-well plates (1 per mutant) BHK 1:_____.

3 days after tranfection:

  1. Aspirate media from fresh plate of BHK cells and wash with PBS.
  2. Transfer media from transfected well to a 2ml tube and centrifuge at 1000rpm and 4C for 2min. Transfer supernatant to 3ml syringe with a 0.2µm filter on the end, and filter media onto corresponding well of fresh plate. Add additional 1.5ml MEM-BHK + 5% FBS to each well
  3. Note: If cells are not prepared, store filtered media at -80C until ready to use
  4. Incubate plate at 36C, watching for CPE. Time of harvest will be dependent upon CPE progression
  • Day 1 after pass
  • Day 2 after pass
  • Day 3 after pass

4. If strong CPE and no indication of residual vaccinia: Collect supernant and pellet cell debris by centrifugation at
1000rpm and 4C for 10min. Transfer supernatant to new tube and use for plaque purification

5. If weak CPE or indications of residual vaccinia: repeat pass (steps 1-3), filtering onto a 60mm dish of BHK.

Current as of 12-15-14

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