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

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:

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:

Coat dishes with gelatin for lentivirus production => cells detach less

Controls

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)