Remove DNA from Bacterial Lysate using PEI

PEI Precipitation

Dissolve 4 g of PEI in ~ 10 ml of 25 mM Tris-HCl buffer (pH: 8.0).

Transfer PEI solution a dialysis tubing (MWCO 3,500 Da)

Dialyze against 1 L of 25 mM Tris-HCl buffer (pH: 8.0) for  24 hrs.

Transfer PEI solution to 15 ml tube. This is 40% stock. Dilute to 10% and store at 4 oC.

Add to Ecoli lysate at 0.1% final:

add dropwise along the walls of the flask containing E coli lysate, while swirling

Spin for 10 minutes at 5000 rpm. or spin for 2 minutes at 14000 rpm.

Collect both pellet and supernatant

Add (NH4)2SO4 to supernatnat to precipitate protein.

Dissolve pellet in Buffer A and load to Ni column

Streptomycin sulfate precipitation

(pET CBD Expression System Manual - NOVAGEN)

Add 50ml 30% (w/v) strept. Sulf.per liter of cell extract. Add the streptomycin sulfate slowly with stirring. After addition, slowly stir the extract at 4°C ON.

Spin the cell extract at 12000g, 4°C, for 30min.

Stir the clarified cell extract for an additional 24 hours at 4°C to complete precipitation.

Once again spin the cell extract at 12000g, 4°C, for 30min to produce a clarified cell extract.