5. Add 15 ml BDIII (3M K+, 5M Acetate pH 4.8; made as follows: 147.3 g K.Acetate, 57.5 ml Acetic Acid, H2O to 500 ml, pH should be ~4.8). Invert sharply several times and incubate on ice for 10 min. Spin in HS4 for 45 min at 7000 rpm.
6. Transfer 21 ml of supernatant to each of two 50 ml polypropylene tubes containing 14 ml isopropanol. Mix and let sit @RT for 5 min. Spin in HS4 rotor for 15 min @ 4.2 K rpm (4800 g) at 4°C.
7. Wash pellet with 70% ethanol. Remove last dregs of ethanol with pipet tip. Dissolve pellets in a total of 8.3 ml LoTE (4 ml per pellet).
8. Weigh 8.8 gm CsCl (Ultrapure) in weigh boat and put into a centrifuge tube. Add exactly 8 ml DNA solution. Density should be 1.57 - 1.60, but you generally do not need to measure this if you have weighed and pipetted out DNA carefully.
9. Add 800 ul 1 mg/ml ethidium bromide in TE.
10. Transfer solution to Ti 70 or Ti 50 ultracentrifuge tubes (both SETON 6041). screw on collars, put tops on. Tighten with screwdriver.
11. Opposite tubes should weigh within 100 mg of each other.
12. Place in Ti 70 rotor and spin @ least 16 (--> 20 hr) @ 50.000 rpm (brake @ 800rpm) @ 20oC.
Alternatively, you can use a Ti 50 rotor and spin at 48 K for the same amount of time.
13. Prepare 2 syringes (3 cc) with 1" 22 gauge needles for each prep. Label syringes so you won't mix them up.
14. Carefully remove tubes from rotor with hemostat.Remove inner nut from centrifuge tubes.
15. Put on face shield. Set up tubes in ring stand above UV transilluminator. Insert needle just above supercoiled band and bevel down to aspirate the band.
16. Pour remaining CsCl in chemical waste and wash centrifuge tube cones and nuts. The plastic centrifuge tubes are disposable but the nuts and cones are not.
17. Extract with an equal volume of H2O saturated 1-butanol in 15 ml polypropylene tube. Saturated butanol = 40 ml butanol + 10 ml H2O; the butanol layer stays on top.
18. Vortex ---> sit 10-60 sec @ RT. Discard top (red) layer
19. Repeat three times (total of 4 extractions). Add equal volume of H2O-saturated butanol each time. (If begin with 1.5 ml, end with ~ 2.5 ml of solution).
20. Add 5 volumes LoTE to final volume of DNA in a 50 ml tube. Vortex, and add 2 volumes of ethanol (e.g., if 2.5 ml DNA, add 12.5 ml LoTE, mix, then add 30 ml ethanol).
21. Centrifuge 60 min @ 4200 rpm (=4800 g).
22. Wash with 75% ethanol twice. Re-spin and remove last drop of 75% ethanol. Do not speedvac.
23. Dissolve in 0.5 ml LoTE.
E. Alkaline Lysis Protocol for Plasmid Minipreps.
1. Pellet 2 ml overnight coli culture in 2-ml eppendorf tubes, and spin for 1 min.
2. Discard the supernatants,
3. Add 200 ul Resuspension Buffer (50mM glucose, 25mM Tris-HCl pH8.0, 10mM EDTA pH8.0), and vortex briefly.
4. Add 200 ul Lysis Solution (0.2N NaOH and 1% SDS), gently mix by inverting the tubes several times.
5. Add 200 ul Precipitation Solution (5M potassium acetate 60 ml, glacial acetic acid 11.5 ml, and water 28.5ml), mix well by inverting the tubes several times,
6. Spin the tubes at top speed for 3 min.
7. Pour the supernatant to a new set of 1.5ml tubes. Add 500ul 2-propanol and mix well.
