Editing Plasmid MaxiPrep using DMAE Column (section)

== Simplified Protocol  ==

<br> All column steps are performed with one full column volume of solution. 

1. Equilibrate column with equilibration buffer (50 mM MOPS 7.0, 500 mM NaCl.). 

2. Resuspend 1-2g cell pellet in 8 ml P1 (50 mM Tris 7.5, 10 mM EDTA, 100 ug/ml RNase A). 

3. In 50 ml conical, lyse with 8ml P2 (200 mM NaOH, 1% SDS) &lt;10min. 

4. Precipitate protein by mixing in 8 ml cold P3 (3M KOAc pH 4.8-5.5). Spin 15,000g 10min. 

5. To remove floating debris, filter the supernatant with coffee paper or cheesecloth.&nbsp;<br> 

6. Add 1 volume of&nbsp; Buffer ENDO-1 to 9 vol of supernatant. Mix.<br> 

7. Apply supernatant to column. 

8. Wash column twice with wash buffer (50 mM MOPS 7.0, 800mM NaCl, 10% ethanol), use&nbsp; Buffer W5-ENDO or Buffer W7-ENDO for endotoxin free plasmid DNA,<br> 

9. Elute with elution buffer (50 mM Tris 7.5, 1.2M NaCl.). 

10. Precipitate DNA with 0.6-1 vol isopropanol. 

11. Wash pellet with 70-80% ethanol, resuspend DNA in buffer of choice. 

12. Regenerate the column by successively applying 1 M NaOH, 2-3 M NaCl, and either water, 

<br> if you will be re-using the column right away, or storage solution (100 mM NaCl, 20%ethanol), if you will be storing the column. Store in a 50 ml conical at 4°.