Editing Chemical Modification of Taq in TMBE (section)

== Purification of Taq ==

Grow Taq-plasmid transformed JM109 in 500 ml TB/amp O/N at 37 oC

Split into 10 L of 1 L of TB/amp, grow to OD600=0.7

Induce with 0.1mM IPTG.

Grow for 26 hours

Harvest cells by centrifugation at 3000×g for 15 min.

Freeze cell pellets

Thaw pellet and resuspend in 25mM Tris–HCl (pH 7.5), 10mM EDTA, 0.2M ammonium sulphate, 50 mg/mL lysozyme

Incubate at room temperature for 15 min

Incubate at 75 ◦C for 1 h toprecipitate heat-sensitive proteins.

Spin at 36,000×g for 30 min.

Add Polyethyleneimine to the clarified cell lysate to a final concentration of 0.1% (v/v)

incubate the mixture on ice with agitation for 1 h to precipitate E. coli host nucleic acids.

Spin at 36,000×g for 30 min to precipitate nucleic acids

Load the supernatant onto a 50mL Phenyl-Sepharose® high substitutionfast flow column (Amersham Biosciences) equilibrated with 25mM Tris–HCl (pH 7.5), 1mM EDTA, 0.2M ammonium sulphate.

Wash column with 3 column volumes of 25 mM Tris–HCl (pH 7.5), 1mM EDTA.

Elute Taq polymerasen with a 0–4 M gradient of urea over 9 column volumes.

Pool fractions containing Taq polymerase.

Dialyse to remove traces of urea

Concentrate using a Vivaflow 50 system in 25mM Tris–HCl (pH 7.5), 1mM EDTA

Store enzyme at 4 ◦C.