Large Scale BAC Prep

Protocol

1. Innoculate a flask containing 500 ml of LB + 12.5 µg/ml Chloramphenicol (Add 184 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.
2. Transfer the culture to a sterile 1 L centrifuge bottle and pellet the cells in the IEC at 2.5k rpm for 20 minutes.
3. Decant the supernatant and resuspend the cells in 24 ml of TES. Transfer the resuspended cells into a square bottom 250 ml polycarbonate centrifuge bottle. 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

4. Add 24 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	--

5. Add 24 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°.

6. Pellet the cell debris by centrifuging in the GSA rotor at 8000 rpm for 15 minutes.
7. Divide the supernatant evenly amoung 3 50 ml conical tubes.
8. Add 8 µl of 10 mg/ml RNase (DNase free) to each tube.
9. Add 20 ml of Phenol/CHCl₃ to each tube. Mix by inverting 10 times. Centrifuge in the IEC at 2.5k rpm for 25 minutes.
10. Combine the divided solutions by transferring the aqueous phase from each 50 ml tube into a 250 ml square bottom polycarbonate centrifuge bottle.
11. Add an equal volume of isopropyl alcohol to the centrifuge bottle. Let the DNA preciptate for at least 1 hour at room temperature.
12. Pellet the DNA in the GSA rotor at 8k rpm for 20 minutes.
13. Decant the supernatant and wash the pellet with 10 ml of 70% EtOH. The pellet may be transferred to a 30 ml Corex tube and centrifuged in the SS-34 rotor at 8k rpm for 10 mintues.
14. Decant the supernatant and dry the pellet under vacuum.
15. Resuspend the pellet in 500 µl of TE and transfer to a 1.5 ml Epindorf tube. Store at 4°.
16. In a typical prep, 4 µl of the resusupended DNA is a reasonable amount digest for running on an agarose gel.