Medium Scale BAC Prep

Protocol

1. Innoculate a flask containing 100 ml of 2XYT + 12.5 µg/ml Chloramphenicol (Add 37 µl of a 34 mg/ml in EtOH stock) with the bacteria containing the BAC you wish to isolate. Grow overnight at 37° with shaking. Note that long growth times increase BAC yield.
2. Transfer half the culture to a sterile Oak Ridge tube and pellet the cells in the SS-34 rotor at 8,000 rpm for 10 minutes.
3. Pour off the supernatant, add the remainder of the culture to the tube and spin again.
4. Decant the supernatant and resuspend the cells in 5 ml of TES. Incubate at room temperature for 5 minutes.

   TES
   [Stock]	Reagent	Amount	[Final]
   1 M	Tris (pH = 7.5)	5 ml	10 mM
   0.5 M	EDTA (pH = 8)	1 ml	1 mM
   5 M	NaCl	10 ml	100 mM
   --	H2O	484 ml	--
   Autoclave

5. Add 5 ml of Lysis Solution. Mix gently but thoroughly by inversion. Incubate on ice for 5 mintues. The mixture should clear as the cells lyse.

   Lysis Solution
   [Stock]	Reagent	Amount	[Final]
   10 N	NaOH	2 ml	200 mM
   20 %	SDS	5 ml	1 %
   --	H2O	93 ml	--

6. Add 5 ml of 3M K, 5M Acetate. Mix gently by inversion. Incubate on ice for at least 5 minutes.

   3M K, 5M Acetate
   Reagent	Amount
   KOAc	29.44 g
   Glacial Acetic Acid	11.5 ml
   H2O	to 100 ml
   Dissolve the KOAc in ~50 ml H2O, add the acetic acid then add H2O to reach 100 ml. Store at -20°.

7. Pellet the cell debris by centrifuging in the SS-34 rotor at 10,000 rpm for 15 minutes.
8. Pour the supernatant into a 50 ml conical tube.
9. Add 5 µl of 10 mg/ml RNase (DNase free) to each tube. Let sit for a while at room temperature or overnight at 4°.
10. Add 15 ml of Phenol/CHCl₃ to the tube. Mix by inverting 10 times. Centrifuge in the IEC at 2.5k rpm for 15 minutes.
11. Transfer the aqueous layer to a 50 ml Erlenmeyer flask.
12. Add an equal volume of isopropyl alcohol to the flask. Let the DNA preciptate for at least 1 hour at room temperature.
13. Transfer the precipitated DNA solution to a 50 Corex tube.
14. Pellet the DNA in the SS-34 rotor at 8,000 rpm for 10 minutes.
15. Decant the supernatant and wash the pellet with 10 ml of 70% EtOH. Transfer the contents of the tube to a 15ml Corex tube and spin at 8,000 rpm for 10 minutes in the SS-34 rotor.
16. Decant the supernatant and dry the pellet under vacuum.
17. Resuspend the pellet in 400 µl of TE and transfer to a 1.5 ml Epindorf tube.
18. Add 400 µl of Phenol/CHCl₃ to the tube. Mix gently by inversion and spin in a microcentrifuge for 10 minutes.
19. Transfer the aqueous phase to a new 1.5 ml Epindorf tube.
20. Add 100 µl of 7.5M NH₄⁺ Acetate to the tube. Mix gently. Add 1 ml of 95% EtOH, mix gently and preciptate at -20° for at least half an hour.
21. Spin the Epindorf tube for 15 minutes at 4° in a microcentrifuge.
22. Decant the supernatant and wash the pellet in 70% EtOH.
23. Dry the pellet under vacuum and resuspend in 100 µl of TE. Store at 4°.
24. In a typical prep, 4 to 6 µl of the resusupended DNA is a reasonable amount to digest for running on an agarose gel.