Editing Restriction Enzyme Purification (section)

== Not1  ==

Grow E. coli ER2169 (NEB# 816) in LB +0.2% Glucose /Amp/Chl to log phase at 37 oC. Add&nbsp; IPTG to a final concentration of 0.5 mM. 

Induce for 2 hours at 37 oC.<br> 

Spin down cells, and freeze at -80 oC. 

All the following procedures should be performed either on ice or at 4° C.<br>1. Resuspend 18 grams of cells in 70 ml of Buffer A 

<br>(10 mM potassium phosphate pH 6.9, 0.1M NaCl, 0.1 mM EDTA, 5% glycerol) 

<br>2. Break cells by freeing-thawing and then homogenization. 

<br>3. Spin at 25000 rpm for 30 min at 4° C. 

<br>4. Load the resulting supernatant onto a phosphocellulose column (2.5×15 cm) equilibrated with buffer A. 

<br>5. Wash the column with 60 ml buffer A and then a 900 ml linear gradient from 0.1M NaCl to 1.0M NaCl. 

<br>enzyme should be eluted at 0.2 to 0.3M NaCl 

<br>6. Pool fractions, concentrate and dialyze against buffer B (0.1M NaCl, 10 mM Tris-HCl pH 7.5, 10 mM MgCl2, 5% glycerol and 0.15% NaAzide) overnight, or buffer exchange using Sephadex G25. 

<br>7. Load onto a Heparin-Sepharose column (1.5×17 cm) equilibrated with buffer B. 

<br>8. Wash the column with 30 ml buffer B, and a 400 ml linear gradient from 0.1M NaCl to 1.2M NaCl in Buffer B. 

<br>The restriction enzyme should be eluted from heparin column between 0.5M and 0.6M NaCl, pool fractions. 

<br>9. Dilute the pooled solution 1:1 with buffer B without NaCl. 

<br>10.Load to a DEAE-Sepharose column (1.5×16 cm) equilibrated with buffer B at 0.3M NaCl. 

<br>The enzyme will not bind and thus should flow through the column. 

<br>11. Collect flow-through diluted 1:1 with buffer B no NaCl, and loaded onto a PEI column (1.5×10 cm) equilibrated with buffer B at 0.15M NaCl. 

<br>12. Wash the column with 20 ml buffer B, and a 300 ml linear gradient from 0.15M to 1.8M NaCl. 

<br>13. The enzyme should be eluted at 0.6M to 0.8M NaCl. 

<br>14. Add BSA (bovine serum albumin) to the pool to a final concentration of 100 μg/ml. 

<br>15. Dialyse the enzyme against storage buffer (10 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 0.1M NaCl, 1 mM DTT, 0.15% Triton-X 100 and 50% v/v glycerol). 

This purification scheme should yield 53,000,000 units of NotI restriction endonuclease<br> 

=== 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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