Large Scale BAC Prep
Based on the protocol from Genome Systems, Inc.
- 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.
- Transfer the culture to a sterile 1 L centrifuge bottle and pellet the cells in the IEC at 2.5k rpm for 20 minutes.
- 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
- 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 —
- 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°.
- Pellet the cell debris by centrifuging in the GSA rotor at 8000 rpm for 15 minutes.
- Divide the supernatant evenly amoung 3 50 ml conical tubes.
- Add 8 µl of 10 mg/ml RNase (DNase free) to each tube.
- 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.
- Combine the divided solutions by transferring the aqueous phase from each 50 ml tube into a 250 ml square bottom polycarbonate centrifuge bottle.
- Add an equal volume of isopropyl alcohol to the centrifuge bottle. Let the DNA preciptate for at least 1 hour at room temperature.
- Pellet the DNA in the GSA rotor at 8k rpm for 20 minutes.
- 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.
- Decant the supernatant and dry the pellet under vacuum.
- Resuspend the pellet in 500 µl of TE and transfer to a 1.5 ml Epindorf tube. Store at 4°.
- In a typical prep, 4 µl of the resusupended DNA is a reasonable amount digest for running on an agarose gel.
This page is, as always, Copyright (c) 1998 by Craig D. Amundsen. All rights reserved.