Cosmid, BACs, PACs and Fosmid DNA Isolation using 96 deep well blocks,
pipetting on the Hydra 96, and growth in the HiGro
Updated 1-25-01 to add shaking in the HiGro and eliminate second acetate pptn.
Latest update 12-12-07 to add the Beckman 96 deep well block number.

  1. Culture Growth:
    1. Pick a smear of colonies and inoculate 5 ml LB medium with the appropriate antibiotic. Grow the culture for 8 hours, shaking at 37 degrees C, 250 rpm to produce the "starter culture". The colonies should be picked and inoculated in the morning, so that the transfer can be done later in the day.
    2. Add 1 ml of LB media containing the appropriate antibiotic to each well of four deep well blocks [Beckman (cat#140504)]. Using a repeat pipettor, transfer 10 ul of the "starter culture" to each well.
    3. Incubate all four inoculated culture containing blocks in the HiGro for 18 hours at 37 degrees C, 520 rpm.

  2. DNA Isolation:
    1. Centrifuge the blocks at 2500 rpm for 8 mins. Discard the supernatant.
    2. Store cells in -20 degrees C freezer for at least two hours.
    3. Add 200 ul of TE-RNaseA/T1 (TE-RNaseA/T1 is: 10 mM Tris-HCl, pH 7.6; 1 mM EDTA; 42 ug/ul RNase A; 0.04 U/ul RNase T1) to each well using the Hydra. Incubate on the table top microtiter shaker at speed of 7 until for 20 minutes to fully resuspended the cells.
    4. Prepare 100 ml of alkaline lysis solution (20 ml of 1N NaOH, 10 ml of 10% SDS, and 70 ml ddH2O to bring the final volume to 100 ml).
    5. Add 200 ul of lysis solution to each well, using the Hydra. Incubate on microtiter shaker at speed of 4 until a clear lysate is developed, usually takes approximately 15 minutes.
    6. Add 200 ul 3M NaOAc, pH 4.5 to each well using the Hydra. Shake in the HiGro at 37degC and a shaker setting of 400 rpm for 30 minutes.
    7. Freeze the blocks at -70 degrees C overnight until totally solid.
    8. Remove the blocks from freezer and thaw. Centrifuge at 4250 rpm for 45 mins in the Jouan KR422 refrigerated centrifuge. At this stage the pellet should be tightly packed and it is critically important that none of the pellet is transfered during the next step or it will result in an unacceptably high level of host (E. coli) genomic DNA contamination of the prep.
    9. Transfer 400 ul of supernatant from each well to new blocks using the Hydra making sure not to transfer any of the pellet or viscous layer immediately above the pellet.
    10. Add 300 ul of isopropanol to each well using the manual Eppendorf repeater pipette.
    11. Centrifuge the blocks at 3000 rpm for 30 mins. in the Beckman CS-6R table top microtiter plate centrifuge to collect DNA. Decant the supernatant. Invert on a paper towel.
    12. Dissolve the pellet by adding 200 ul of 10:1 TE to each well, using the Hydra followed by mixing by shaking on the table top microtiter shaker at speed of 7 for 20 minutes to fully dissolve the pellet. Move to step 14 below as the double acetate precipition given in step 13 has been eliminated (but is included for historical reasons).
    13. The following double acetate steps can be eliminated.
      1. Add 100 ul of 7.5 M of KOAc** to each well, using the manual Eppendorf repeater pipette.
      2. Freeze the blocks in -70 degrees C for approximately until solid (one hour to overnight).
      3. Thaw. Centrifuge at 2500 rpm for 30 mins. in the Beckman CS-6R table top microtiter plate centrifuge.
      4. Transfer 250 ul of DNA from each well to a new block, using the Hydra.
      5. Add 600 ul of 100% ethanol, using the manual Eppendorf repeater pipette.
      6. Centrifuge at 3000 rpm for 30 mins. in the Beckman CS-6R table top microtiter place centrifuge. Decant.
      7. Wash by adding 1 ml of 70% ethanol to each well, using the manual Eppendorf repeater pipette. Centrifuge at 3000 rpm for 10 mins. in the Beckman CS-6R table top microtiter place centrifuge. Decant
      8. Invert on a paper towel. Vacuum dry for 30 mins. Move on to step 18 below.
    14. Add 500 ul of 95% ethanol-acetate to each well, using the manual Eppendorf repeater pipette.
    15. Centrifuge at 3000 rpm for 30 mins. in the Beckman CS-6R table top microtiter plate centrifuge. Decant.
    16. Wash each pellet with 500 ul of 70% ethanol and centrifuge immediately at 3000 rpm for 10 mins. in the Beckman CS-6R table top microtiter plate centrifuge. Decant and drain by inverting on a paper towel.
    17. Vacuum dry for 30 minutes (no longer as additional time makes the pellet difficult to dissolve.
    18. Dissolve the DNA in each well in 20 ul of sterile ddH2O, using the Hydra followed by mixing by shaking on the table top microtiter shaker at speed of 7 for 20 minutes to fully dissolve the pellet.
    19. Centrifuge in the Beckman CS-6R table top microtiter plate centrifuge for approximately 20 seconds to collect the liquid in the bottom of the wells.
    20. Incubate overnight at 4 deg C to completely dissolve the DNA before proceeding.
    21. Pool the samples in groups of 12 with the 12 channel pipettor into one set of 12 wells. Then, transfer the DNA solution from these 12 pooled wells equally into one orange cap, 15 ml, V-bottom Corning Tube #430788.
    22. The typical yield is ~500 ug from 4 blocks of starting BAC or Cosmid culture.
    23. Prepare a regular agarose gel to test DNA. Load 10 ul samples on gel with the MW markers and electrophores at 50-60 mA.
    24. After determining the yield and concentration, if needed, the DNA can be concentrated by room temperature precipitation with 2 volumes of 95% ethanol-acetate, centrifugation, briefly drying and dissolving in a lower volume of water.
**7.5 M KOAc - 736.13 gm/liter final volume - Starting with a small volume of ddwater (approximately150ml) slowly add the dry potassium acetate and mix as it is added. Then add additional water and the remainder of the acetate until all is dissolved and the final volume is 1 liter. DO NOT ADJUST THE pH as the acetate will come out of solution.

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Bruce Roe, broe@ou.edu