Team:MIT mge

From 2010.igem.org

mammalian genetic engineering protocol

The Mammalian team created lines of cells using lentiviruses for transfection.

1 Calcium Phosphate Transfection
    1.1 Preparation
    1.2 Protocol
2 Filter and Concentrate Virus
    2.1 Protocol
3 Materials Needed
    3.1 HBSS Buffer
    3.2 Calcium Chloride Solution
    3.3 Freezing medium
    3.4 Supplemented DMEM

Calcium Phosphate Transfection

Beforehand:
  • Combine CaCL2 and dilute Na2PO4 to get CaPO4 with DNA in it
    • Particles must be of the right size for cells to take them up, controlled by pH: exactly 7.06
    • Use each aliquot 1-2 times then throw away because air contact makes the pH drift.
  • To make lentiviruses, need:
    • Gagpol for packaging (on PDR)
    • membrane prot on pVSV-G
    • Desired DNA
  • Cells should be at 50-70% confluence
  • Seed cells the day before: For 150 mm dish, seed about 7million cells
  • Can start with just confluent dish:
    • Split cells into 2 (to get about 50%)
    • 30 mins for cells to adhere (5-6h to spread out)
  • Coat dishes with gelatin for lentivirus production => cells detach less
  • Controls
    • No cells (see precipitate, like fine snow on cells)
    • Just carrier DNA (any plasmid)

Protocol
  • 1. Mix DNA (different plasmids in right amounts) with CaCl2
  • 2. Quickly vortex in 14 mL tube
  • 3. Add water to ~1.5 mL total volume
  • 4. Must saturate water with air - vortex without cap for 20-30 sec
  • 5. Make 1.5 mL HBSS aliquot
  • 6. For each tfxn, we now have 1 tube HBSS and 1 tube DNA+CaCl2+H2O in 14 mL tubes
  • 7. Add DNA solution dropwise to HBSS - 10-20 sec total - while vortexing solution. Use 2mL pipette for adding
  • 8. Immediately suck up into 5mL pipette and stop reaction by loading onto cells (spread by pipetting onto medium, no need to shake
  • 9. Check precipitation under microscope (small black dots between cells)
  • 10. Put cells back into incubator
  • 11. Next morning, change medium
  • 12. 1 and again 2 days after: Harvest 20mL (i.e. all the medium) to filter and centrifuge to obtain virus.

Filter and Concentrate Virus

Protocol
  • 1. Start with 20 + 20 mL culture supernatant
  • 2. Ultracentrifuge tubes (kept on shelf in tissue culture room) may contain residual bleach
    • Wash 2x with 70% EtOH
    • Wash 1x with medium
  • 3. Filter through 0.45 um (0.2 um can be used) Corning vacuum filter into 50 mL tube into ultracentrifuge tube
  • 4. Everything that contacts the virus including tips and plates must be bleached
  • 5. Spray filters with 10% bleach
  • 6. After filtered supernatant, add to each tube 4-5 mL sterile 20% w/v sucrose in PBS solution
    • Purpose of sucrose is to remove excess VSVG protein and dampen impact of particles
    • Phases will mix a little
  • 7. Balance weight with PBS to within <5mg
  • 8. Ultracentrifugation
    • Speed: 45,000 RCF (g)
    • Rotof: JA 25.50 (max 8 tubes)
    • Time: 3h
    • Accel: slow
    • Decel: OFF
    • Temp: 4°C
  • 9. When finished, cannot wait too long or pellet detaches
  • 10. Mark pellet, then aspirate supernatant
  • 11. Resuspend in 100uL PBS (or HBSS) for total of ~150 uL

Materials

HBSS Buffer (500 mL)
    • 8.0g NaCl
    • 0.37g KCl
    • 106.5mg Na2HPO4 (anhydrous, 201.1 mg if 7x H2O)
    • 1.0g dextrose
    • 5.0g Hepes
  • Add all components into a beaker and add 400 mL of Nanopure/bidistilled water. Dissolve and adjust pH exactly to 7.06 (wait 20 min. to be sure that the pH won't change anymore).
  • Filter through a 0.2 um filter, aliquot into 15 mL plastic tubes (12mL/tube). Can be kept at -20 for a few months.

Calcium Chloride Solution (50mL)
  • 5M CaCl2 (18.4g CaCl2*2H2O in 50mL)
  • Keep at +4°C

Freezing medium
  • 12mL FCS
  • 15mL DMEM (no antibiotics, directly as supplied)
  • 3mL DMSO
  • TOTAL: 30mL

Supplemented DMEM
  • 450mL DMEM +Glucose +glutamine
  • 50mL FBS
  • 5mL Pen/Strep
  • 0.5mL Fungin
  • TOTAL: 500mL

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