Qiagen MaxiPrep

Anion-Exchange Chromatography using Qiagen MaxiPrep Column

1. Resuspend E. coli cells in XX mL Buffer P1.

2. Add xx mL Buffer P2, mix the solution, and incubate at room temperature for 5 min.

3. Add xx mL Buffer P3 and mix carefully.

4. Incubate the lysate for 30 min at room temperature (15–25 oC) to allow the flaky white precipitate of SDS, protein, genomic DNA, and cell residue to rise to the surface.

5. Carefully pump the lysate out of the bottle.

6. Mix the lysate with 0.10 vol of buffer ER (QIAGEN) and filter the mixture through a QIAfilter unit (QIAGEN). Collect the filtrate for subsequent chromatography.

7. Equilibrate the QIAGEN Ultrapure 100 Column with 350 mL buffer QBT.

8. Load the column with the filtrate from step 6.

9. Wash the charged column with  Buffer QC.

10. Elute plasmid DNA with  Buffer QN.

11. Precipitate the DNA with 0.7 vol of isopropanol at 4 oC and centrifuge at 6000 rpm for 10 min at 4 oC.

12. Wash the DNA pellet with  75% ethanol.

13. Dry the DNA pellet and resuspend it in the appropriate buffer for further applications.