Medium Scale BAC Prep
Based on a protocol from Genome Systems, Inc.
- 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.
- 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.
- Pour off the supernatant, add the remainder of the culture to the tube and spin again.
- 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
- 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 —
- 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°.
- Pellet the cell debris by centrifuging in the SS-34 rotor at 10,000 rpm for 15 minutes.
- Pour the supernatant into a 50 ml conical tube.
- 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°.
- 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.
- Transfer the aqueous layer to a 50 ml Erlenmeyer flask.
- Add an equal volume of isopropyl alcohol to the flask. Let the DNA preciptate for at least 1 hour at room temperature.
- Transfer the precipitated DNA solution to a 50 Corex tube.
- Pellet the DNA in the SS-34 rotor at 8,000 rpm for 10 minutes.
- 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.
- Decant the supernatant and dry the pellet under vacuum.
- Resuspend the pellet in 400 µl of TE and transfer to a 1.5 ml Epindorf tube.
- Add 400 µl of Phenol/CHCl3 to the tube. Mix gently by inversion and spin in a microcentrifuge for 10 minutes.
- Transfer the aqueous phase to a new 1.5 ml Epindorf tube.
- 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.
- Spin the Epindorf tube for 15 minutes at 4° in a microcentrifuge.
- Decant the supernatant and wash the pellet in 70% EtOH.
- Dry the pellet under vacuum and resuspend in 100 µl of TE. Store at 4°.
- In a typical prep, 4 to 6 µl of the resusupended DNA is a reasonable amount to digest for running on an agarose gel.
This page is, as always, Copyright (c) 1999 by Craig D. Amundsen. All rights reserved.