Editing Restriction Enzyme Purification (section)

=== QC  ===

<br>The NotI restriction endonuclease obtained from this purification method should be substantially pure and free of non-specific endonuclease and exonuclease. 

1), Ligation: After a 25-fold overdigestion of Adeno2 DNA, greater than 95% of the DNA fragments produced can be ligated with T4 DNA Ligase (at a 5' termini concentration of 1-2 uM at 16° C.). Of these ligated fragments, 95% should be able to be recut. 

2), Prolonged digestion: After incubating a 50μl reaction containing 1 μg of Adeno2 DNA and 200 units of enzyme for 16 hours, the same pattern of DNA bands should be produced as a reaction performed in one hour with one unit of enzyme. 

3), Exonuclease Activity: After incubation of 200 units of enzyme for 4 hours at 37° C. in a 50μl reaction containing 1 μg sonicated 3 H DNA (105 cpm/μg) less than 0.03% radioactivity should be released. 

4), Endonuclease Contamination: After incubation of 200 units of enzyme for 4 hours at 37° C. in a 50μl reaction containing 1 μg φX174 RFI DNA, less than 5% should be converted to RF II. 

5), Megabase genomic Digest: Digestion of one microgram of E. coli genomic DNA embedded in 0.5% agarose incubated with 200 u NotI for 16 hours in 100μl NEBuffer#3 resulted in the same limit digest banding pattern as 5 u in 4 hours, as determined by PFG electrophoresis. 

All tests can be performed in the following reaction buffer: 

NEBuffer 3 (100 mM NaCl , 50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT (pH 7.9 at 25° C.) supplemented with 100 μg/ml BSA.<br> 

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