CsCl EZ Band Cleanup System
Protocol
Revision No. 2008-999-5K02W

For Rapid and Environmentally-Correct Elimination of Ethidium Bromide from supercoiled DNA CsCl Bands

How it works:

1. The supercoiled DNA band from the CsCl gradient is passed over a special silica matrix. The ethidium bromide and some of the DNA binds to the cartridge.
2. The DNA that bound to the cartridge in 1 is eluted and pooled with the DNA that did not bind.
3. Purified DNA is precipitated and dissolved at the desired concentration and is ready for any subsequent reaction or use.

1. Pre-wet cartridge with 4 ml of Cartridge Loading Solution.



2. Load ethidium bromide (EtBr)-stained DNA from CsCl Gradient onto the cartridge (directly from the syringe used to remove it from the ultracentrifuge tube). Be certain to collect all flow-through that is expelled by pushing 2 syringe-volumes of air through cartridge. The cartridge will bind all ethidium bromide and up to 3 mg of DNA.



3. Wash column with 4 ml of CsCl Wash #1* (A dramatic elution of the EtBr will occur).




*Add equal volume of 100% ethanol to CsCl Wash No. 1 before first use.




4. Repeat cartridge wash with 4 ml of CsCl Wash #1.



5. Wash cartridge with 4 ml of CsCl Wash #2** .




**Add 45 ml (12 prep kit)/90 ml (24 prep kit)/180 ml (50 prep kit) of 100% ethanol to CsCl Wash No. 2 before first use.




6. Elute DNA with 10 ml CsCl Elution Solution using a slow even-flow rate; collect flow-through into a 50 ml centrifuge tube and add flow-through from step 2.



7. Add 500 µl of 5M NaCl and 9 ml of room temperature isopropanol. Invert 3-4 times to mix thoroughly. Incubate at -70°C for 20 minutes and centrifuge at 15,000 x g for 15 minutes at 4°C (e.g., 11,000 rpm in an SS-34 rotor; 10,000 rpm in an HB-4 swinging bucket rotor), and carefully decant supernatant. (Isopropanol pellets are somewhat transparent and more difficult to see than salt-containing ethanol DNA pellets. To assist in locating pellet, mark the tube prior to centrifugation when using a fixed angle rotor).



8. Carefully rinse pellet with 80% ethanol and aspirate off all residual liquid. [If pellet dislodges, centrifuge at 15,000 x g for 10 minutes at 4°C and carefully decant supernatant. Pulse spin (i.e. ~3000 rpm < 1 min in a tabletop centrifuge) and remove any residual ethanol with an aspirator. Air dry for 5-10 minutes at room temperature. Resuspend pellet in 0.5-1.0 ml TE or H₂0 (be sure to recover the DNA which might be smeared along the wall of the tube above the pellet when using a fixed angle rotor). Store DNA at 4°C. [If you choose to resuspend the pellet in H₂0, add 1% TE when measuring concentration by OD to obtain an accurate OD260/280 reading].



9. Centrifuge cartridge in tabletop centrifuge in a 50 ml conical tube with 2-3 Kimwipes packed at the bottom to absorb solution. Spin for 5 minutes at 1000 rpm (Beckman GPR swinging bucket rotor). [Longer centrifugation will not be harmful; the purpose of this step is to eliminate residual EtOH. A vacuum manifold may also be employed].