Team:MIT mge

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Mammalian Protocol

mammalian microfluidic protocol

The Mammalian team focused on creating a pressure-sensitive promoter and creating a standard protocol for mammalian genes.

Mammalian Genetic Engineering Protocol

Before Calcium Phosphate Transfection:

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:

Calcium Phosphate Transfection

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.

PDMS Baking

1. Bake the poured wafers for 4-8 hours at 80C (recommended: 24hours to cross-link most of PDMS monomers)
2. Detach by cutting with razor (carefully and slowly peel it off, starting from the edges and going in the circumferential direction, in order to avoid tearing posts apart)