Paul.xu: Created page with "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-N..."

2012-03-07T15:05:50Z

Created page with "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-N..."

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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]]

Plasmid GigaPrep using FPLC - Revision history

Paul.xu: Created page with "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-N..."