BACDNAIsolationfrom200mlCulturesbyaClearedLysateMethod

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.

6、The supernatant is transferred into one 250 ml bottle per 200 ml 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 GSA 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 3.6 ml of 10:50 TE (10 mM Tris-HCl, pH 7.6, 50 mM EDTA, pH 8.0), and 1.8 ml of 7.5 M KOAc (note: the 7.5 M KOAc solution is used without pH adjustment) is added into each bottle and transfered to a 50 ml Sorval centrifuge tube.

8、After mixing, the tubes were frozen, until solid, at -70 degrees C. This takes at least 30 minutes but typically was frozen overnight.

9、The following ribonuclease treatment step reduces the amount of RNA present in the final BAC preparation. After thawing, centrifuge at 10K for 10 minutes in the SS34rotor. Then, 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. The DNA is precipitated by adding 30 ml of cold 95% ethanol 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 centrifuge. Each pellet then is washed with 30 ml of 70% ethanol and dried in vacuum.

10、Each pellet is dissolved in 210 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.

11、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.

12、The DNA concentration then is estimated by analysis of 10 ul of the 210 ul final DNA solution by agarose gel electrophoresis and by measuring the A260 in the spectrometer. Typical BAC yields per 200 ml of original cell growth is approximately 150 ug.