CsCl-Ethidium Bromide Gradient Plasmid Preparations

Method:

1. Grow E. coli overnight in 500 mls media that selects for the presence of plasmid.
2. Spin down cells at 8000 rpms for 5 minutes in G3 rotor. Drain thoroughly.
3. Resuspend pellet in 3 mls of ice-cold 25% sucrose in 0.25 M Tris, pH 7.0. Transfer to a 10 ml ultracentifuge tube.
4. Add 0.6 mls of freshly prepared 30 mg/ml lysozyme in H₂0. Mix. Place on ice for 5 minutes.
5. Add 1.2 mls of ice-cold 0.25 M EDTA, pH 8.0. Mix. Place on ice for 5 minutes.
6. Add 1.5 mls of 5 M NaCl. Mix. Immediately add 0.6 mls of 10% SDS. Mix. Leave on ice for 3-4 hours.
7. Pellet lysed cells at 40,000 rpm in a 50Ti rotor at 4^oC in the ultracentrifuge for 45 minutes.
8. Pour supernatent into a 12 ml falcon tube, leaving behind the last bit of viscous clear supernatent (mainly chromosomal DNA). Bring volume to 7.50 mls with sterile water.
9. Add exactly 6.5 grams of CsCl. Mix (wrist action) until dissolved.
10. Add 1 ml of 10 mg/ml Ethidium Bromide (EtBr). Mix. (Caution: EtBr is a mutagen. Wear gloves. Use care. 30 µg = 1 cigarette).
11. Transfer solution to a Beckman quickseal tube (50Ti). Using a 16 gauge needle and a syringe simplifies this. Overlay with parrafin oil. Seal.
12. Spin 48-60 hours at 44,000 RPM in a 50 Ti rotor or 70.1 Ti.
13. Collect the plasmid (lower) band after removing the chromosomal (upper) band. Setup the tube in a clamp over a collection bucket with lots of K-dry around. Wear gloves and a lab coat (EtBr). Visualize with LONG-WAVE UV lamp. Wear a face shield. Collection is done by first puncturing the very top of the tube to allow air in. Then puncuture the tube (3cc syringe with 18 gauge needle) right above the top band and lower the tip into band and suck it off. After all the very viscous portion of the chromosomal band has been removed sick the needle in the opposite wall and get a new 3cc syringe and 18G needle. Stick it in above the plasmid band and suck it off. This is the stuff you want for step 14. Cleanup. (see Maratz (New) p. E8)
14. Extract the EtBr from the plasmid by the addition of an equal part of NaCl-saturated isopropanal (mix equal parts of 5 M NaCl and isopropanol, let sit until the salt settles to the bottom of the tube and there are two phases, use the top phase). Mix. let sit and remove top phase. Repeat until the top phase becomes clear. Do it one more time.
15. Transfer to sterile corex tube that will hold ten times the volume you’ve got. Add two parts water. Mix. Add two parts (new volume, 6 parts original volume) of -20^o C 100% ethanol. Mix. Leave overnight at -20^oC (no longer).
16. Spin down in SS34 (with adaptors) at 8,000RPM for 20 minutes at -20^oC. Drain pellet. Dry. Resuspend in 400 µl of TE or water. Transfer to a 1.5 ml microfuge tube and phenol extract once. Add 60 µl of 3 M Sodium acetate. Mix. Add 800 µl of cold Ethanol. Mix. Leave in -20^oC for a few hours or on dry ice for 10 minutes. Spin down in microfuge in cold room for 15 minutes. Pour off supernatent. Wash with -20^oC 70% Ethanol and spin for 5 minutes. Pour off supernatent and dry pellet in vacuum. Release vacuum slowly (through a paper towel). resuspend in 100 µl TE and take the A₂₆₀, A₂₈₀ and A₃₁₀ of a 1/200 dilution of your prep. 1 A₂₆₀ unit = 50 µg/ml. A₂₆₀/A₂₈₀ ratio should be between 1.5 and 2.0. A₃₁₀ should be very near zero. Alternatively, quantitate with Hoefer TRO₁₀₀ Fluorometer H33228^P dye. (See manufacturer’s method in this manual)