BAC DNA PREP

Modified for our equipment from Joe Takahashi's protocol.

This works fine so far on Bacs up to 150 kb....

1. Grow up 200ml culture (LB 12.5 ug/ml Chloramphenicol) for 16 hrs. 300 rpm 37oC)

2. Collect the cells by centrifugation in 250 ml polypropylene centrifuge tubes in the J10 or J14 rotor 15 min 6000 rpm. (about 6,000 x g) Drain supernatant.

3. Resuspend by adding 20 mls P1 buffer (buffer recipes from Qiagen) and make sure there are no clumps. Pipetting better than vortexing? Transfer now to 2 Oakridge tubes or transfer clumpy snot later.

4. Add 20 mls of P2 and INVERT GENTLY to mix. Incubate for << 5 min. (Solution should clarify)

5. Add 20 mls of P3 and INVERT GENTLY to mix. Incubate on ice 10-15 min.

6. Transfer suspension to 2 40ml Oakridge tubes (if you haven't already) and clear lysate by centrifugation at 12,000 rpm for 25 minutes at 4oC in the J20 rotor.

7. Transfer the supernatants by pouring through cheesecloth (key step). To 4 orange cap 50ml conicals (~15 ml each). Bring volumes up to 50mls with 100% Ethanol (cold) and centrifuge at 3000 rpm for 15 minutes in the tabletop centrifuge. (Lino has one with conical supports so your tubes don't split open!) Should be a fat pellet.

8. Discard supernatant making sure to get rid of anything white, globular and still floating in solution. (this is more cell debris and protein which should not appear after the clearing spin above).

9. Add 20mls 70% Ethanol (cold) vortex and centrifuge at 3000 rpm for 5 minutes in the tabletop.

10. Drain supernatant. Invert to dry for 5 minutes. Dry under vacuum briefly, as in stand there and watch them.

11. Dissolve DNA pellet in 4 ml final vol. of TE containing 10-20 ug/ml RNAse A. Usually goes into solution immediately. Allow to resuspend overnight? Or press ahead after 30 min at 37oC.

12. Add 4.64 grams of Cesium Chloride per 4 ml of TE suspended pellet. Cap and carefully invert to dissolve CsCl. After dissolving add 400 ul of Ethidium Bromide (10mg/ml) and invert to mix (EtBr will initially settle on top... Make sure it distributes throughout solution). Warning: This is a known mutagen. Handle with CARE! Dispose of wastein appropriately designated EtBr waste.

13. Centrifuge samples at 3000 rpm for 10 minutes in the tabletop centrifuge. Transfer supernatant avoiding the proteinaceous debris to new 15ml polypropylene tubes. At this point weigh 100 ul of the solution to determine its density. Add CsCl or TE empirically to end up with a solution of density 1.55 g/ml.

14. Once density is adjusted, load all the solution into 5.3 ml vTi65 ultracentrifuge tubes. Should just fill tube. Seal the tubes and place in the vTi65 rotor, tighten cap holder with torque wrench.

15. Centrifuge samples at 58,000 rpm in Beckman ultracentrifuge for 16-20 hrs at 20-25oC.

16. View bands under long wavelength UV source. (keep this to a minimum exposure) Vent the tube by piercing with needle. Use the syringe with an 18 gauge needle to pierce the tube and collect the lowest band (supercoiled BAC DNA). Be careful to pierce a few mm below the band and aim slightly upwards with bevel positioned upwards just beneath the band. Carefully draw out solution.

17. Extract with water-saturated n-butanol to remove Ethidium bromide. Pipet off the butanol (top layer) and replace between extractions. Continue until the solution is not pink and then do it again one final time.

18. Dialyze against TE (1 Liter) with 2 changes for > 24 hrs. Room temp is ok. I do this to avoid ethanol ppt and problems with final resuspension. Remember this DNA is for injection and must be clump-free! I think the dialysis is actually the key step in the procedure.

(as modified from Wash U. protocol by LW/AS May 6, 1998 and by CF from LW/AS 9/97)