DNA Isolation by a Cleared Lysate Method Followed by Double Acetate Precipitation Version 3d - updated July 3, 2001

Adapted from the Genome Sequencing Center, Washington Univ. Protocol

  1. A smear of colonies of cosmid/BAC/PAC were picked and transferred into a 12X75 mm Falcon tube containing 3 ml of LB medium (10 g Bacto-Tryptone, 5 g Bacto-yeast extract, and 10 g NaCl in 1 L H2O, autoclaved) with appropriate antibiotic. After incubating at 37 degrees C for 8-10 hours with 250 rpm shaking, the culture is transferred to a 250 ml flask containing 50 ml of the same medium and incubated for 8-10 hours under the same conditions. Then, the 53 ml of the culture is divided into 4X 2 liter flasks containing 1 liter of the same medium and incubated for additional 8-10 hours. After harvesting the cells by centrifugation at 5K rpm for 15 minutes in a 500 ml bottle in the RC5-B using the GS3 rotor, cell pellets should be frozen and stored at -70 degrees C.
  2. Prior to use, the cells from 1l growth were thawed and resuspended in 40 ml of just 10 mM of EDTA, pH 8.0 by gently pipetting up and down with a 10 ml pipette do not vortex. Cells should be resuspended completely. The cells suspend more efficiently with 10 mM EDTA alone (rather than with GET 50 mM glucose 25 mM Tris-HCl, pH 8.0 and 10 mM of EDTA, pH 8.0). After mixing gently, the solution is incubated at room temperature for 5 minutes.
  3. To resuspended cells, 80 ml of alkaline lysis solution (0.2 N NaOH and 1% SDS) is added and after very gently swirling until the solution is homogenous, it is incubated for 5 minutes at RT. The whole step should be finished within 10 minutes.
  4. Immediately, 60 ml of cold, approximately 3 M KOAc (made by mixing 50 ml of 7.5 M KOAc with 23 ml of HOAc and 127 ml of ddH2O, stored at 4 degrees C) is added and mixed very gently by swirling the bottle several times and then the bottle is placed in a freezer (either -20 or -80 deg C) and stored frozen at -20 or -80 deg C overnight. Freezing at this stage recently is shown to result in a much cleaner final DNA preparation as the resulting pellet is much more tightly packed if this samples are frozen for several hours prior to clearing the lysate by the subsequent centrifugation step.
  5. The lysate is cleared from precipitated SDS, proteins, membranes, and chromosomal DNA by centrifuging at 10,000 rpm for 15 minutes at 4 degrees C in the RC5-B using the GSA rotor. Prior to re-centrifugation, the cleared lystate supernatant can be filtered through a double-layer of cheesecloth to remove any floating material. Then, an additional centrifugation is performed to ensure that all insolubles were removed.
  6. The supernatant is transferred into one 500 ml bottle per liter of original cell growth and then an equal volume of isopropanol is added and mixed by swirling. After centrifugation at 5,000 rpm for 15 minutes in RC5-B using GS3 rotor, the supernatant is decanted and the pellet drained.
  7. The second acetate precipitation step is performed by quickly and gently dissolving the DNA pellet in 18 ml of 10:50 TE (10 mM Tris-HCl, pH 7.6, 50 mM EDTA, pH 8.0), and 9 ml of 7.5 M KOAc (note: the 7.5 M KOAc solution is used without pH adjustment) is added into each bottle.
  8. After mixing, the bottles were frozen at -70 degrees C for at least 30 minutes or overnight.
  9. The following method is prefered over the alternative given below as it results in less RNA present in the final BAC (or cosmid or PAC) preparation. After the solution is thawed and centrifuged at 6,000 rpm for 10 minutes at 4 degrees C in the RC5-B using the GSA rotor, the supernatant of each tube is transfered into 50 ml Corning centrifuge tube and DNase-free RNase A is added to a final concentration of 100 ug/ml from a 20 mg/ml stock and RNase T1 is added to a final concentration of 40 ul/100 ml from a 100 U/ul stock followed by incubation in a 37 deg C water bath for 1 hour. Then, 30 ml of cold 95% ethanol is added into each tube. After mixing, by inverting, the tubes were incubated in an ice-water bath for 15 minutes. The DNA then is pelleted by centrifugation at 3000 rpm for 25-30 minutes in a Beckman GS-R centribuge. Each pellet then is washed with 30 ml of 70% ethanol and dried in vacuum.
  10. Alternatively, after the solution is thawed and centrifuged at 6,000 rpm for 10 minutes at 4 degrees C in the RC5-B using the GSA rotor, the supernatant is precipitated with 2.5 volumes of ethanol. After centrifugation at 8,000 rpm for 15 minutes at 4 degrees C to precipitate the DNA, the pellet is resuspended in 1.4 ml of 50:50 TE (50 mM Tris-HCl, pH 7.6, 50 mM EDTA, pH 8.0). and once dissolved, 20 ul of 20 mg/ml DNase-free RNase A is added, followed by incubation at 37 degrees C with shaking at 250 rpm for 30 minutes. Then the solution is divided equally into two eppendorf tubes (per liter of original growth) and 700 ul of phenol is added to each tube and gently mixed followed by centrifugation for 5 minutes at 12,000 rpm at room temperature. Each supernatant is transferred to another clean tube and extracted with 700 ul of phenol again. The final supernatant then is extracted with 700 ul of ether by vortexing gently and centrifuging as above. The phenol extracted DNA in the aqueous phase is combined with 700 ul of isopropanol, mixed gently and centrifuged for 5 minutes at 12,000 rpm at room temperature. The DNA pellet finally is washed with 500 ul of 70% ethanol and after collection by centrifugation as above, it is dried in the vacuum chamber for 5-10 minutes.
  11. Each pellet is resuspended in 500-1000 ul of 10/0.1 TE by pipetting up and down and incubating at 37 degrees C followed by storing at 4 degrees C overnight to ensure all the high molecular weight large insert clone DNA will be dissolved completely.
  12. Note: If end sequencing this large insert clone, then a portion should be re-precipitated with ethanol and dissolved in ddH20 instead of TE since the EDTA inhibits Taq Polymerase.

    Typical yields per liter of original cell growth range from 2.5 mg for cosmids to 0.5 mg for BACs. The DNA concentration then is estimated by agarose gel electrophoresis and by measuring the A260 in the spectrometer.

    Note: RNase digestion could be done at other steps, for example, it could be done before adding isopropanol, before adding KOAc, or after DNA is dissolved in ddH2O.

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    Bruce Roe, broe@ou.edu