Editing Restriction Enzyme Purification (section)

== Kpn1  ==

1. Suspend cells in Buffer A 10 mM potassium phosphate (pH 7·0), 1 mM EDTA, 7 mM b-mercaptoethanol. and lyse cells by freezing-thawing and homogenization. 

2. Spin at 25000 rpm for 30 min. 

3. Collect supernatant. <br>

Optional add 1% polyethyleneimine (PEI) or&nbsp;freshly made 10% (w/v) streptomycin sulfate was added to a final concentration of 1 %. <br> in the presence of 250 mM NaCl. Spin again for 15 min to precipitate DNA. Then add ammonium sulphate to 50% for fractionation. Spin to and collect pellet. Dissolve pellet in 5 ml Buffer A and dialyze against buffer A o/n. 

4. Load sample to a 5 ml Hi-Trap heparin column or large scale 70 ml&nbsp; column volume, wash and elute the enzyme with Buffer A containing 2–0·8 M NaCl. Combine the fractions containing the enzyme (between 200–350 mM NaCl), dialyse against Buffer B 20 mM Tris-HCl, pH 7·4, 0·1 mM EDTA, 50 mM NaCl, 5 mM b-mercaptoethanol. 

5. Load to Mono-Q (HR1O/10) column and eluted with a linear gradient from 0.1 M to 0.5 M NaCl in Buffer B.<br>

6. Pool fractions containing Kpn1 and dialyse against storage buffer, 10 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 0.2 M KCl, 1 mM DTT, and 50 % glycerol.<br><br>50 milligrams of KpnI can be purified from 12 g of E. coli AH29. The molecular weight (Mr) of KpnI is <br>approximately 31,000 daltons.<br><br>

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