@For the purification of supercoiled plasmid DNA which is of maximum purity & is suitable for use with any molecular biological enzymes, the use of CsCl/ethidium bromide is clearly the method of choice. It is, however, relatively expensive in reagent costs (CsCl), materials (disposable centrifuge tubes) and centrifuge time. The time saved working with messy DNA is generally worth it, however.
@In general, amplification of the plasmid DNA is not required, & is not used in this protocol.
- Reagents
- STE
| 100mM |
NaCl |
| 50mM |
Tris, pH 8 |
| 1mM |
EDTA |
- Lytic mix
| 10% (w/v) |
sucrose |
| 10mM |
Tris, pH 8 |
| 5mM |
EDTA |
- YT
| 8g/l |
tryptone |
| 4g/l |
yeast extract |
| 4g/l |
NaCl |
- Lysozyme sol'n
@10mg/ml lysozyme in lytic mix (prepare fresh)
- Triton sol'n
| 2% |
triton X-100 |
| 50mM |
Tris, pH 8 |
- CsCl/EtBr sol'n
dissolve for each ml of STE/CsCl, add 80ul 10mg/ml Ethidium Br
- Saturated 2-PrOH
Mix equal volumes of:
- isopropyl alcohol
- 5M NaCl, 50mM Tris, pH8, 1mM EDTA
Shake vigorously, & allow to settle. Use the upper, organic phase.
- TE
- Methods
- Prepare an overnight culture of the plasmid-bearing strain from which the plasmid will be isolated by inoculating 5-10ml of YT with a drop of a previous culture or a loop-full of cells scrapped from a plate. Incubate overnight at 37C with shaking.
- Inoculate 1liter YT with 1-2ml of the overnight culture & incubate overnight again at 37C with vigorous shaking.
- Harvest the cells by low-speed centrifugation (i.e. 5KRPM, 5 min., 4C in an HB4 or GSA rotor) and resuspend the cell pellet in 50ml STE. Repellet 5KRPM, 5 min., 4C in an SS34 rotor and resuspend the cell pellet in 4.5ml ice-cold lytic sol'n.
- Add 0.9ml lysozyme sol'n, mix in, & incubate on ice for 5 min.
- Add 1.8ml 250mM EDTA, mix in, & incubate on ice for 5 min.
- Add 7.2ml Triton sol'n, mix in, & incubate on ice for 20 min. If the suspension does not become viscous, incubate at 37C for 5 min., & recool on ice.
- Transfer the resulting goo to Ti60 tubes & spin 35KRPM, 1hr, 4C (or use an SS34 at 18KRPM for 1 hr at 4C if you can't get a Ti60). Collect the supernatant (the pellet should resemble an oyster, & have the same rubbery consistency) & measure it's volume.
- For each ml of cleared lysate, add 1g CsCl. Dissolve gently (don't kick up alot of foam). Remeasure the volume, & for each ml add 80ul 10mg/ml ethidium bromide.
- Centrifuge 10KRPM (SS34 or HB4 rotor in Sorval) in Corex tubes. Recover the CsCl/EtBr/extract solution from underneath the EtBr/protein/junk pellicle.
- Divide into tubes for the Ti75, Ti60, VTi60 or VTi65, top off with CsCl/EtBr sol'n (or add 3.5ml @ to SW50 rotor tubes, fill & balance with mineral oil) & close the tubes (except SW rotors) with caps or heat-sealer. Load the tubes into the rotor & centrifuge as specified:
| Rotor |
Time |
Temp |
Rotation |
| VTi60, VTi65 |
ON |
20C |
45KRPM |
| Ti75, Ti60 |
2days |
20C |
40KRPM |
| SW50, SW50.1 |
ON |
20C |
35KRPM |
- Using a short-wave UV light to visualize the DNA bands, collect the lower, plasmid DNA bands by syringe through the sides of the tubes (don't forget to puncture the top of the tube if its heat-sealed, or open if it's capped, before inserting the syringe). If you have lots of DNA, the band should be easily visible without need for UV illumination.
- If using a vertical rotor, pool the samples & transfer to a fresh centrifuge tube. Top off with CsCl/EtBr sol'n & centrifuge as before. Once again, collect the plasmid DNA band via syringe through the side of the tube.
- Mix with an approximately equal volume of Sat'd 2-PrOH, & allow to settle for a few minutes. Collect & discard the upper, 2-PrOH phase. Repeat this until both phases are colorless, then repeat twice more for good measure.
- Dialyse overnight with 2 changes of TE at 4‘C.
- Store at 4C. Examine the DNA on a minigel & extimate the concentration either by UV spectrophotometry (A260) or using an EtBr spot test.
Maniatis, Mol. Cloning Manual
Davis, Mol Cloning Manual
Davis Cram, personal communication
Greg Beckler, personal communication