BAC DNA Isolation using 96 deep well blocks, pipetting on the Hydra 96, and growth in the HiGro

Fares Z. Najar, Mandi Aycock, Doris M. Kupfer and B. A. Roe
11-25-03 and updated on 03-09-06 for pEpiFOS-5 induction
and on 12-12-07 to add the Beckman 96 deep well block number

A. Culture Growth:
  1. For BAC isolation for shotgun library construction:
  2. For BAC or Fosmid isolation for end sequencing:
B. DNA Isolation:
  1. Centrifuge the blocks at 3000 rpm for 10 mins. Discard the supernatant.
  2. *DO NOT FREEZE* the cell pellets but continue with the next steps in the isolation without pausing.
  3. Add 300 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 (The Hydra will add 300 ul by adding 150 ul twice).
  4. Incubate on the table top microtiter shaker at speed of 7 until for 20 minutes to fully resuspended the cells.
  5. Add 300 ul of lysis buffer (1% SDS, and 0.2 N NaOH) to each well, using the Hydra. Incubate on microtiter shaker at speed of 4 until a clear lysate is developed, takes approximately 15 minutes.
  6. Add 300 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.
  8. Remove the blocks from freezer and thaw. Centrifuge at 3200 rpm for 45 mins in a Beckman CS-6R table top microtiter plate centrifuge.
  9. Transfer 500 ul of supernatant from each well to new blocks using the Hydra (The Hydra will transfer 500 ul by transferring 250 ul twice).
  10. Add 500 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 40 ul of steril double distilled water (instead of 10 ul of 10:1 TE to each well which should be added if the BAC or Fosmid DNA is to be used for shotgun cloning) using the Hydra followed by shaking on the table top for 20-30 minutes to dissolve DNA.
  13. For isolating BACs and Fosmids for end sequencing skip to the last step, i.e. electrophores an alaquot of the dissolved DNA on a 1% agarose gel as the second acetate cleanup step is not necessary. Typically 10 ul of this 40 ul DNA solution is used for each end sequencing reaction with 1:8 Big Dyes or Amersham ET mixes, 2 ul of universal primer from a 6.5 umolar primer stock solution, in a final volume of 14 ul followed by 60 cycles of cycle sequencing amplification and loading onto the ABI 3730xl.
  14. For BAC or Fosmid DNA isolation for shotgun sequencing:
  15. Run 1% agarose gel for qualitative assessment.

C. Shearing and cloning
  1. Shear 50-100 ug DNA with the Hydroshear (Gene Machines, Inc) set to generate 2-4 Kb size fragments.
  2. Precipitate the nebulized DNA using EtOH-OAc and wash with 70% EtOH
  3. Resuspend in 20 ul of dd-water.
  4. Fill-in and kinase treat the sheared DNA according to the standard protocol.
  5. Run a low-melt agarose gel for size selection.
  6. After excising the desired size range, extract DNA using the "freeze and squeeze" method with 2 rounds of freezing or the ultrafree-DA centrifugal filters from Millipore.
  7. Precipitate the DNA using EtOH-OAc and wash with 70% EtOH.
  8. Dissolve the DNA in 6 ul dd-water and use the entire solution for ligation and electroporation into pUC18 using the standard protocol.

Notes:
**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