LargeScalePlasmid,Cosmid,BAC,PAC,andFosmidDNAIsolation

DNA Isolation by a Cleared Lysate Method Followed by Double Acetate Precipitation Version 3b - updated September 26, 1999

The Most Recent Roe Lab Implementation (by Feng Chen, Hau-Qin Pan and Fu Ying) Adapted from the Genome Sequencing Center, Washington Univ. Protocol

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 was 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 was 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.

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 was incubated at room temperature for 5 minutes.

To resuspended cells, 80 ml of alkaline lysis solution (0.2 N NaOH and 1% SDS) was added and after very gently swirling until the solution was homogenous, it was incubated for 5 minutes at RT. The whole step should be finished within 10 minutes.

Immediately, 60 ml of approximately cold 2 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) was added and mixed very gently by swirling the bottle several times and then the bottle was placed in an ice-water bath for at least 5 minutes. The pellet also can be stored frozen at -20 deg C and then centrifuged at a later date. Freezing at this stage recently was 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.

The lysate was 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. Then an additional centrifugation was performed to ensure that all insolubles were removed.

The supernatant was transferred into one 500 ml bottle per liter of original cell growth and then an equal volume of isopropanol was added and mixed by swirling. After centrifugation at 5,000 rpm for 15 minutes in RC5-B using GS3 rotor, the supernatant was decanted and the pellet drained.

The second acetate precipitation step was 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) was added into each bottle.

After mixing, the bottles were kept at -70 degrees C for 30 minutes. After the solution was thawed and centrifuged at 6,000 rpm for 10 minutes at 4 degrees C in the RC5-B using the GSA rotor, the supernatant was 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 was resuspended in 1.4 ml of 50:50 TE (50 mM Tris-HCl, pH 7.6, 50 mM EDTA, pH 8.0).

Once dissolved, 20 ul of 20 mg/ml DNase-free RNase A was 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 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.

Each pellet is resuspended in 500 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. 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.

DNA Isolation by a Cleared Lysate Method Followed by Double Acetate Precipitation Version 2

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 was transferred to a 250 ml flask containing 50 ml of the same medium and incubated for 8-10 hours under the same conditions. Then, all 53 ml of the culture was transferred into a 2 liter flask containing 1 liter same medium and incubated for additional 8-10 hours. After harvesting the cells by centrifugation at 7,000 rpm for 20 minutes in a 500 ml bottle in the RC5-B using the GS3 rotor, cell pellets were frozen and stored at -70 degrees C.

Prior to use, the cells were thawed and resuspended in 35 ml of GET (50 mM glucose, 25 mM Tris-HCl, pH 8.0, and 10 mM of EDTA, pH 8.0) by gently pipetting up and down. Cells should be resuspended completely. Then 70 mg of lysozyme was added to a final lysozyme concentration of 2 mg/ml. After mixing gently, to make sure lysozyme was dissolved completely, the solution was incubated at room temperature for 10 minutes.

To resuspended cells, 70 ml of alkaline lysis solution (0.2 N NaOH and 1% SDS) was added and after very gently swirling until the solution was homogenous, it was incubated for 5 minutes in an ice-water bath.The whole step should be finished within 10 minutes.

Immediately, 52.5 ml of 3 M NaOAc, pH 4.8 was added and mixed very gently by swirling the bottle several times and then the bottle was placed in an ice-water bath for no more than 15 minutes. The lysate was cleared from precipitated SDS, proteins, membranes, and chromosomal DNA by pouring through a double-layer of cheesecloth, transferring the lysate into 250 ml centrifuge bottle and centrifuging at 10,000 rpm for 30 minutes at 4 degrees C in the RC5-B using the GSA rotor. If necessary, an additional centrifugation was performed to ensure that all insolubles were removed.

The supernatant was transferred into a 500 ml bottle. An equal volume of isopropanol was added and mixed (inverting the bottle), the solution was held at room temperature for 5 minutes prior to centrifugation at 9,000 rpm for 20 minutes in RC5-B using GS3 rotor. The supernatant was decanted and the pellet drained.

The DNA pellet was quickly and gently dissolved in 18 ml of 10:1 TE (10 mM Tris-HCl, pH 7.6, 1 mM of EDTA, pH 8.0), divided into two 50 ml Corning centrifuge tubes and 4.5 ml of 7.5 M KOAc (note: the pH of the 7.5 M KOAc was not adjusted) was added into each tube. After mixing, the tubes were kept at -70 degrees C for 30 minutes. After the solution was thawed and centrifuged in the Beckman GS-6R centrifuge at 2,000 rpm for 10 minutes, the supernatant of each tube was transferred into 50 ml Corning centrifuge tube and DNase-free RNase A was added to a final concentration of 100 ug/ml, followed by incubation in 37 degrees C water bath for 1 hour. Then 30 ml of 100% cold ethanol was added into each tube. After mixing by inverting, the tubes were incubated in an ice-water bath for 15 minutes. DNA pellet was formed by spinning at 3,000 rpm for 25 minutes in Beckman GS-6R centrifuge. The pellet in each tube was washed with 30 ml of 70% ethanol and dried in vacuum oven. The dried pellet usually was dissolved in a total of 2 ml of ddH₂O and the DNA concentration was estimated by agarose gel electrophoresis.

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

Still need to add biomek/robbins automated protocols and eventually end up with 3 protocols using Biomek or Robbins or some combination of each:

1. Automated plasmid isolation of sequencing templates.

2. Automated cosmid,bac,pac, etc isolation for shotgun cloning.

3. Automated cosmid,bac,pac, etc isolation for sequencing directly from.