BAC DNA Isolation from 200 ml Cultures by a Cleared Lysate Method

1、A smear (rather than a single colony) of BAC colonies are 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 500 ml flask containing 200 ml of the same medium and incubated for 8-10 hours under the same conditions. After harvesting the cells by centrifugation at 5K rpm for 15 minutes in a 250 ml bottle in the RC5-B using the GSA rotor, cell pellets should be frozen and stored at -70 degrees C.

2、Prior to use, the cells from 200 ml growth were thawed and resuspended in 8 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, 16 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, 12 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.