CsCl CIRCLEPREP^® Protocol
Step Gradient Kit for Plasmid Purification
Revision No. 2009-999-6B01W

For reference only; see Detailed Protocol below

1. Resuspend 4 g cell pellet in 50 ml water.
2. Add 4 ml of CsCl Alkaline Lysis Solution, (part I) and 3 ml of part II; gently invert 15 times.
3. Add 30 ml of CsCl Neutralization Solution; mix, and pellet debris.
4. Add 60 ml isopropyl alcohol and centrifuge to pellet crude DNA/RNA mixture.
5. Dissolve pellet in water, RNase, isopropanol precipitate; and resuspend pellet in CsCl DNA Solution.

1. Adjust volume and dissolve 4.0 g of Crystalline DNase-Free CsCl.
2. Add Ethidium Bromide Solution; mix; centrifuge, and discard pellet.
3. Add to an ultracentrifuge tube and fill with less dense CsCl Solution.
4. Centrifuge overnight at 45,000 rpm.

1. Remove lower plasmid DNA band from gradient with syringe and needle.
2. Pass through EZ-Band Cleanup Cartridge.
3. Washaway ethidium bromide with CsCl Wash #1.
4. Wash with CsCl Wash #2.
5. Elute plasmid DNA; precipitate, and dissolve in CsCl Elution Solution.

The protocol and prep size is based on a 13 ml ultracentrifuge tube and centrifugation in a fixed angle rotor, e.g., (Beckman Ti80, Ti60). Other tubes and rotors can be used with the same procedure by making appropriate volume and ultracentrifuge time modifications.

1. Pellet cultured cells in a 1 liter or 250 ml centrifuge bottle(s); resuspend cell pellet(s) (maximum 4 g wet weight of packed cells/prep) in a total of 50 ml of water (by vortexing, pipetting, and/or vigorous shaking by hand) until a homogeneous suspension is achieved. Cell suspension should be placed in a single bottle before continuing.
2. Add 4 ml of CsCl Alkaline Lysis Solution part I and 3 ml of CsCl Alkaline Lysis Solution part II. Gently invert 15 times. The solution will become fairly clear and highly viscous as lysis occurs.
3. Add 30 ml of ice cold CsCl Neutralization Solution; shake up and down 5-8 times vigorously by hand. The precipitate that forms as small flakes will slowly rise to the surface of the liquid. Centrifuge at 6000 x g for 2 minutes and pour the supernatant through the Sieve Material (placed in a funnel) into a clean container. (Clean Sieve Material)
4. Add 60 ml of room-temperature isopropanol; mix; and centrifuge at 10,000 x g for 5 minutes to pellet DNA/RNA precipitate. Dissolve/resuspend (pellet may not completely dissolve and will instead be a cloudy suspension; see option below) pellet in 10 ml of CsCl DNA Solution and transfer to a 50 ml centrifuge tube. If the suspension is cloudy, it can be spun again to eliminate the insoluble debris; the DNA will stay in the supernatant. Add 40 µl of RNase Mixx. Incubate at room temperature for 15 minutes.
5. Add 10 ml of isopropanol; mix and centrifuge at 10,000 x g for 5 minutes to pellet isopropanol-precipitated DNA. Discard supernatant and place tube upside down on a paper towel to drain residual isopropanol. Dissolve pellet in 1.5 ml CsCl DNA Solution.

Instructions are for 13 ml gradients. For different volume gradients, adjust reagent volumes accordingly. Time Consideration: Approximately 30 minutes for setup and overnight centrifugation in fixed angle rotor.

1. Adjust volume to 2.5 ml with CsCl DNA Solution and add 4 g of DNase-Free Crystalline CsCl. Mix until CsCl dissolves.
2. Add 400 µl of Ethidium Bromide Solution (wear gloves and use proper precautions when working with ethidium bromide); mix, and centrifuge to pellet precipitated debris; some will pellet, some will float.
3. Add supernatant to a 13 ml polyallomer ultracentrifuge tube (avoid transferring particles of precipitate. Discard and treat precipitate in proper manner as it contains ethidium bromide). Slowly and carefully layer approximately 7 ml of less dense CsCl Liquid Solution onto the more dense DNA/ethidium bromide-containing solution until tube is full. Alternatively, add 7 ml of the less dense CsCl Liquid Solution to an empty centrifuge tube and layer the denser DNA-containing CsCl Solution under the lighter solution with a Pasteur pipet.
4. Cap or seal full tube and centrifuge at 25°C overnight (16 hours or longer in a fixed angle rotor) at 45,000 rpm (in a Ti60 rotor, for example; Do not exceed this speed. Do not use a rotor that has a derated maximum of <55,000 rpm). Stop rotor without brake.

Remove lower plasmid DNA band from gradient with syringe and needle: If the quantity of DNA in the band is 0.5 mg or more, it will be visible in room light. If necessary, use a longwave UV light to guide removal of DNA band: Loosen cap to equalize pressure inside and outside of tube and remove DNA from gradient by puncturing the side of the tube with an 18 gauge needle on a syringe at the lower edge of the band and slowly withdraw the band. Alternatively, with cap removed, withdraw and discard CsCl above the plasmid band with a pipet and, with a new pipet, remove the plasmid band. Avoid upper, non-supercoiled DNA band.

Continue from this point with the CsCl EZ Band Cleanup System Protocol.