Editing Plasmid GigaPrep using FPLC

1. Grow a single colony of bacteria in 5 ml LB.

2. Grow bacteria in a Wave Bioreactor or a Bioflo 3000 bioreactor.

3. Spin down bacteria.

4. Lyse bacteria using Alkaline SDS-NaOH method. 

5. Clarify the lysate with centrifugation and depth filtration.

6. Ultrafilter it 10- to 15-fold using a hollow-fiber cartridge (GE Healthcare) with a 30-kDa nominal molecular-weight cut-off (NMWC). 

7. Pass concentrated solution through a bed of USP-grade Celite diatomaceous earth from Celpure (Sigma Aldrich) to reduce endotoxin content.

8. Separated pDNA from bacterial RNA and other impurities using size-exclusion column chromatography with Sepharose 6 Fast Flow resin (GE Healthcare) in the presence of 2M ammonium sulfate (JT Baker).

9. Collect the first peak containing plasmid DNA.

10. Pass solution through a second Celite bed to further reduce endotoxin. 

11. Ultrafilter and diafilter into Tris-EDTA buffer.<br>

[[Category:Protocols]]