BAC and Fosmid DNA Isolation using 96 deep well, blocks, pipetting on the Hydra 96, and growth in the HiGro Shaker for 454 sequencing

 

As modified by Faras Najar

Roe Lab, University of Oklahoma

Updated September 1, 2008

 

  1. Pick a smear of colonies and inoculate 1 ml TB medium containing the appropriate antibiotic.

 

  1. Grow the culture overnight at 37 degrees C by shaking at 250 rpm in the Floor Shaker to produce a "starter culture".  For multiple projects, this step can be accomplished using the 96-deep well block [Beckman (cat#140504)].

 

  1. Add 1 ml of TB medium containing the appropriate antibiotic to each well in a 96 deep well block [Beckman (cat#140504)].

 

  1. Inoculate 2 ul of the starter culture into each well using the Eppendorf repeater pipette or the Hydra if the starter culture is in a 96-deep well block.

 

  1. Grow the BAC clone for 24 hours at 37 degrees C shaking at 500 rpm in the HiGro.

 

  1. Centrifuge the blocks at 3000 rpm for 10 minutes. Discard the supernatant.

 

  1. Immediately, 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).

 

  1. Incubate on the table top microtiter shaker at speed of 7 until for 20 minutes to fully resuspended the cells.

 

  1. Using the Hydra, 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.

 

  1. Add 300 ul 3M KOAc, pH 4.5 to each well using the Hydra.

 

  1. Seal the block with Acetate Plate Sealer (Dynex, cat.#3501) and mix by holding the block between the  palms of your hands and GENTLY rock the block back and forth several times.

 

  1. Freeze the blocks at -70 degrees C overnight.

 

  1. Remove the blocks from freezer and thaw. Centrifuge at 4250 rpm for 45 mins in the Jouan KR422.

 

  1. Transfer 500 ul of the supernatant from each well to a new block using the Hydra

 

  1. Add 500 ul of isopropanol to each well using the manual Eppendorf repeater pipette.

 

  1. Centrifuge at 4250 rpm for 45 mins in the Jouan KR422 to collect DNA. Decant the supernatant. Invert on a paper towel.

 

  1. Wash with 70% ethanol, Centrifuge at 3000 rpm for 10 mins in the Jouan KR422, and air-dry for 10 minutes.

 

  1. Dissolve the pellet in 50 ul of 10:50 TE (10 mM Tris-HCl, pH 7.6, 50 mM EDTA, pH 8.0) using the Hydra followed by shaking on a table top microtiter shaker at speed of 5 for 20-30 minutes to dissolve DNA.

 

  1. Add half a volume (25 ul) of 7.5 M of KOAc using Eppendorf repeater pipette and  Freeze at -80 degrees C overnight.

 

  1. Remove the blocks from the freezer and thaw.  Centrifuge at 4250 rpm for 45 mins in the Jouan KR422.

 

  1. Transfer 50 ul of the supernatant using the Hydra or the 12-channel pipette.

 

  1. Add 50 ul of isopropanol and Centrifuge at 4250 rpm for 45 mins in the Jouan KR422.

 

  1. Wash with 70% ethanol twice and dissolve the DNA in 20 ul sdd-water.

 

Notes:

** 7.5 M KOAc - 736.13 gm/liter final volume - Starting with a small volume of dd-water (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 precipitate.

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Bruce Roe, broe@ou.edu