Genomic DNA isolation from fixed cells

Resuspend 1-3 X10⁶ cells in 250µL of PBS and transfer to a 2 mL tube (this would represent one 3.5 cm dish of 3T3 cells).

Add 200µg Proteinase K (from a 20mg/mL stock) and 200µg RNase A (from a 20mg/mL stock).

Incubate cells at 37°C for 0h 30m 0s in a water bath.

Add 250µL Qiagen AL lysis buffer per 250µL of the protease and RNAase-containing cell suspension and mix thoroughly.

Place tubes in an incubator at 56°C with shaking at 800rpm,0h 0m 0s , capped.

Add 250µL, 100% molecular biology grade ethanol; mix slowly using a slow vortex for 0h 0m 5s.

With a razor blade, trim the tip of a 1mL pipet tip to enlarge the opening. Use this tip to pipet out DNA from the ethanol solution and apply it onto a silica spin DNA binding column (e.g. EconoSpin 1920-250).

Spin at 6000x g,0h 0m 0s for 0h 1m 0s in a fixed angle tabletop microfuge; aspirate and discard flow-through.

Add 500µL Qiagen buffer AW1 to the column, spin again at 6000x g,0h 0m 0s for 0h 1m 0s, aspirate and discard flow-through.

Add 500µL Qiagen buffer AW2 , spin at 8000x g,0h 0m 0s for 0h 1m 0s, aspirate and discard flow-through.

Spin once more using microfuge to remove excess ethanol at 13000x g,0h 0m 0s for 0h 1m 0s.

Transfer column into a new, 1.5 mL collection tube.

Elute with pre-warmed, 100µL nuclease-free water or TE .

Incubate for 0h 1m 0s, then spin at 13000x g,0h 0m 0s for 0h 1m 0s.

Add another 50µL nuclease free water to accomplish a second elution.

Incubate at Room temperature for 0h 1m 0s, then spin as before at 13000x g,0h 0m 0s for 0h 1m 0s.

The two flow-through fractions contain the genomic DNA .

Perform Nanodrop and Qubit HS DNA estimation to calculate yield.