Posted on June 2nd, 2016 by UNM CC
M. Fero — 3/2003
Most plasmids can be adequately prepped by kits containing DNA binding columns. These columns do not do a great job of separating plasmid DNA from contaminating bacterial chromosomal DNA. If the chromosomal DNA is not sheared most of it will pellet along with the bacterial cell wall. However the relative purity of the plasmid DNA will drop if the plasmid is large or at a relatively low copy number in the prep. The combination of a kit prep with cesium banding eliminates this problem by selective purification of supercoiled plasmid DNA in a equilibrium gradient. The elimination of nicked circular plasmid DNA and bacterial cell wall endotoxins is important to avoid inducing cell cycle checkpoint arrest and toxicity during transient transfections of mammalian cells.
- Grow 250 - 500 mL of bacteria overnight in LB with 50 µg/mL of ampicillin.
- Transfer culture to Nalgene bottles. Spin in SGA rotor at 6000 rpm, at 4ºC for 10 min.
- Purify the plasmid according to the Qiagen Maxi or Mega prep protocol.
- Resuspend the DNA in 3.7 mL of T.E.
- Add 3.7 gm. CsCl and 0.37 mL of 1 mg/mL ethidium bromide.
- Check the density of the DNA solusion by placing 0.01 mL (= V) on an analytical balance and recording its weight (g = Mo). If the solution is too dense add more TE by the following formula: ∆V= (M - 1.55V)/0.55 [∆V is the amount of TE to add in mL]
- Transfer the solution to an ultracentrifuge tube and top off with Cesium Chloride solution (d = 1.55).
- Heat seal the centrifuge tube.
- Spin for 4 hours in a Ti65 vertical rotor at 65,000 RPM, at 20ºC, no brake, or overnight at 55K RPM.
- Remove the lower (supercoiled DNA) band through a syringe and needle with UV illumination. Using UV illumination is important to be aid the visualization both bands. The upper band is nicked or linear plasmid DNA or bacterial chromasomal DNA and must be avoided.
- q.s. to 4 mL with T.E. in a 15 mL corex tube.
- Add 400 µL 3M NaOAc and then 8.8 mL cold EtOH, mix. Incubate at -20ºƒC, 30 min.
- Spin in a swinging bucket rotor at 10,000 RPM, 10 min.
- Wash with 70% EtOH x2. (short spins may be needed if the pellet is loose).
- Air dry and then resuspend in 0.5 - 1 mL TE
- Qiagen Maxi Prep kit
- TE, pH8
- Cesium Chloride
- CsCl/TE solution (d = 1.55 g/mL) 30 gm CsCl + 33 mL TE, check density
- Ethidium bromide 1 mg/mL
- Absolute ethanol
- 70% ethanol
- 3M NaOAc