Team:MIT mge

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Mammalian Protocol

mammalian genetic engineering protocol

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

Calcium Phosphate Transfection

Beforehand:

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:

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

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

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.