Large Scale Plasmid Prep for Low Copy Plasmids
Qiagen Midi Columns
- If you want to purify low copy number plasmids, I end up growing 250ml or larger cultures. Spin down the cells in 250ml bottles and for each bottle resuspend in 30ml of Solution I. Then add 250ul 10mg/ml RNaseI (again the cheap stuff) per bottle.
- Then, on ice, 60ml of Solution II followed by 50ml Solution III.
- Spin and precipitate supernatant with isopropanol.
- Resuspend each pellet in 5 mls of 50mM MOPS pH7.0, 750mM NaCl, 2mmEDTA (this is what Qiagen recommends for resuspending DNA made by other procedures than their own--I don't use their directions for DNA preps unless its pUC or Bluescript or some other high copy number plasmid--the EDTA I added for extra measure.)
- I then add another 100ul of RNaseI for good measure and let sit for awhile (10minutes to an hour) at RT. The DNA suspension will be slightly cloudy, so I spin it down in a clinical (everything done in corning disposable tubes) and put the clarified supernatant on their column and then follow their directions for washing and eluting.
- The final eluted volume is a bit of a pain in the butt (5mls) but it is easy enough to precipitate in another 15ml disposable tube and spin down in the clinical. You have to trust that the DNA is on the side of the tube as you won't see a pellet. Dump off the sup, buzz spin, take off rest of alcohol with pipetteman and resuspend pellet in 500ul TE.
- Reprecipitate in eppendorf with 1ml ethanol and resuspend pellet in TE to whatever final volume you want.
I know it sounds like a lot of work but it is still much faster than CsCl. If you want to purify pUC or SK follow their directions. My prep has worked well for pMMB and pDN derivatives (IncP and IncQ plasmids), although you get a lot of SS DNA from pMMB. No way around it as the column binds DNA and RNA of any shape or form. Thats why I am so anal about adding RNase. Let me know if you want more details.
|Solution I||Solution II||Solution III|
|5 mM sucrose|
10 mM EDTA
25 mM Tris, pH 8.0
|0.2 N NaOH |
1% (w/v) SDS
|3 M sodium acetate,|