Editing PEI Precipitation (section)

== Remove DNA/RNA from Bacterial lysate <br> ==

=== PEI precipitation<br> ===

Determine the amount of PEI necessary to clarify your lysate by performing a titration of PEI on a series of aliquots of your sample. Add 0-140 µl/ml 10% PEI (Polymin P of Sigma) to each aliquot and vortex briefly.<br>

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Add 1 ml to&nbsp; 4 ml of 10% PEI to 100 ml of&nbsp;''E coli'' extract containing 4% (dry weight) nucleic acids. Final PEI concentration is 0.1%.&nbsp; This will almost completely removes nucleic acids. '''Buffer pH should be 7.5-8.0''', at this pH PEI is completely cationic and nucleic acids are fully anionic.

<br>Usually 0.08% to 0.1% w/v is enough for a 10 mg protein/ml solution at pH 7.5 to 8.0.

Spin for 2 minutes at 14000 rpm.

Load 5µg samples of each supernatant in PAGE-SDS gel and stain with Coomasie Blue.

The maximum level of PEI that does not precipitate your target protein can be applied to the entire protein sample. '''''Add PEI slowly, with stirring''''', until the target PEI % is reached. Stir for an additional ten minutes.

Clarify by centrifugation at 10000g for 10min.

Decant supernatant. The lysate can be further purified by '''affinity chromatography (e.g, load to Ni column)''' or, if desired ammonium sulfate precipitation.<br> <br> 

=== Streptomycin sulfate precipitation<br>  ===

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.<br> <br>