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Use filter tips, double gloves, bleach waste, GMO desinfectant, desinfect all plastic material before disposal.

Day 1 - HEK replate

  1. Re-seed HEK cells to obtain 10-20% confluence (ideally early in the morning)
  2. After HEK adhesion (late afternoon) proceed with transfection using either Fugene or Lipofectamine 3000

Day 1A - Transfection by Fugene

  1. Confirm plasmids concentration and prepare the mix of packaging plasmids
  • plasmid #9: 1750ng
  • plasmid #10: 3240ng
  • plasmid of interest: 5000ng.
  1. Equlibrate Optimem to RT
  2. Vortex and spin down Fugene
  3. Dilute plasmid mix in 1mL Optimem, vortex, spin down
  4. Add 30uL of Fugene, mix by inverstion incubate 5-15minutes at RT
  5. Apply dropwise to 10cm dish
  6. Incubate overnight

Day 1B - Transfection by Lipofectamine

  1. Confirm plasmids concentration and prepare the mix of packaging plasmids
  • plasmid #9: 1750ng
  • plasmid #10: 3240ng
  • plasmid of interest: 5000ng.
  1. Equlibrate Optimem and Lipofectamine components to RT
  2. Vortex and spin down components
  3. Solution A: Dilute 35uL of Lipofectamine in 875uL of Optimem, vortex, spin
  4. Solution B: Dilute 20uL of P3000 from Lipofect kit in 875uL of Optimem, add your plasmids, mix by pipetting
  5. Mix A and B, incubate 10-15 min at RT
  6. Apply dropwise to 10cm dish
  7. Incubate overnight

Day 2 - Exchange media

Change media to 10 %KOSR in DMEM high Glucose (15mL)

Day 3-5 collect media with viral particles

  1. Collect media
  2. Put fresh 10 %KOSR in DMEM high Glucose (15mL)
  3. SPin down debris in collected media using 3000g for 10min
  4. Filter using 0.45um filter
  5. Store at 4°C

Day 5 Concentrate particles

NOTE: may not be needed for well transducable cells such as HEK, Cal51

  1. Use Vivaspin Concentrator spin columns
  2. Centrifuge particles at 3000g at 4°C for 15 min
  3. Check if they are concentrated enough if not repeat (I concentrate 20-40x for CMs)
  4. Store concentrated at 4°C for up to 1 month

Transduction test

  1. Seed your cells in 24 well
  2. Test ratio of viral particles / media in range 4to1 to 0.25to1
  3. Check after 24hours what concnetration works best

Transduction

  1. Prepare culture media with Polybrene 5-10ug/mL stock 10mg/mL 7.5ug/mL works good(VV)
  2. Add particles dropwise to wells
  3. Check after 24 hours

Plasmids

  • Packaging #9: Addgene #12259; Stored in Sofia’s freezer top right drawer
  • Packaging #10: Addgene#12260; Stored in Sofia’s freezer top right drawer
  • YAP1-OE #124: Addgene#74942; in Vladmir’s green box in IPSc

Material

Transfection reagents (IPSCs fridge)

Collection and concentration

  • 50mL syringe
  • 0.45um filters
  • Concentration columns Vivaspin 20PES, 100kDa; in western-blot cabinet bottom shelf, the one with blue tops

Transduction

  • Polybrene Sigma Aldrich: 10mg/mL; in cell culture freezer, upper right mbd drawer in 50mL falcon (20F1 B2)