CSCL-PLASMID PREP

Day before plasmid prep:

Innoculate a 10 ml culture at noon and use this to innoculate a 500ml culture in a 2l flask p.m.

Book the Sorvall centrifuges for a 3 to 4 hour block

Book the ultra-centrifuge and VTi65 rotor for two consecutive nights, overnight.

Make up Soln C and chill O/N

I. Alkaline lysis

1. Pellet bacteria in 500 ml bottle at 5000 rpm in GS-3 rotor for 20 min. at 4° C.

2. Resuspend cells thoroughly by pipetting to a total of 8 ml in GTE: 50 mM glucose, 25 mMTris-Cl, pH 8.0, 10 mM EDTA). Transfer the cells to 50ml centrifuge tubes (round bottom, blue snap on lid)

3. Add 2 ml of 25mg/ml lysozyme in GTE. Mix well. RT for 5 min.

4. Add 20 ml of freshly made & chilled solution II (0.2N NaOH, 1% SDS). Cap and flick tubes to mix. Ice for 10 min.

5. Add 15 ml of an ice-cold Soln C Cap and mix by inverting sharply several times. Ice for 10 min. At this point, you will observe a "curds and whey" material.

6. Centrifuge in an SW34 at 18,000 rpm for 25 min. at 4°C. The chromosomal DNA and bacterial membrane should form a relatively tight pellet on the bottom of the tube.

7. Divide the supn in to two 40 ml screw-cap tubes. Avoid bits of pellet .

8. Add 0.6 volumes of isopropanol to each tube mix well and stand at room temp for 15 min t to precipitate DNA.

9. Pellet DNA at 10,000 rpm for 20 min in SW34 at RT -- lower temperatures may result in RNA precipitation.

10. Wash pellet with 70% EtOH and re-spin for 5-10 min.

11. Dry pellet in tissue culture hood with blower on, and UV light OFF. Lay the tubes on their side, with the mouth resting on the blower grid. They should dry within 30 min.

12. Resuspend each pellet by adding 2 ml TE at 55oC for ~ 15-30 min. Combine tubes and adjust vol to exactly 4 ml.

Cesium Chloride Gradients

13. To each 1 ml of TE/DNA add 1.1 gram of cesium chloride. (For instance, to the above example, add 4.4 grams of cesium chloride. Accuracy at this step is crucial.) Let the tubes stand for about 20 minutes or so at this step to make sure everything is dissolved. Add 400l of ethidium bromide 10mgs/ml.

14. Excess fuzzy purplish aggregates may be spun out at 3000 rpm in the Beckman tabletop centrifuge, for 10 min. Check the density of your CsCl soln, as this is crucial to recovery of DNA. This can be done with the departmental refractometer. The refractive index of the solution should be between 1.384 and 1.388, with the ideal density falling at 1.386.

15. Transfer the supernatant to 5 ml quickseal tubes, topping off with 1g/ml CsCl as needed. NOTE: The CsCl is made up by adding 1 ml TE for every gram of CsCl. In this way, 10 grams of CsCl + 10 ml of TE = approx 13 ml total. Do not bring 10 grams of CsCl up to 10 ml -- this solution will be too dense. After sealing the tubes, centrifuge at 55,000 rpm O/N (or for a minimum of 8 hrs) in the VTi65 rotor.

16. After removing the tubes from the rotor, you should observe two bands in the gradient -- the bottom one contains your supercoiled DNA. The top one is nicked DNA. Carefully collect the bottom band using a syringe and an 18 guage (or larger) needle. Add CsCL/Ethidium bromide to each prep, and re-band, as in step 3.

17. Extract Ethidium as described in Maniatis and then dialyse O/N into TE - NB CsCl is dense and high salt- leave room in the sac for expansion of volume & fasten sacs to top of measuring cylinder or they will sink and get caught on the stir bar.

19. Take OD260 and calculate concentration of DNA as follows: (50) x (OD) x (dilution factor) /1000 = ug/ul

Soln III -- 5 M KAc, pH 4.8

100 ml 500 ml

5M Potassium Acetate 60 ml 300 ml

glacial acetic acid 11.5 ml 57.5ml

dH2O 28.5 ml 142.5 ml