U.S. Dept Commerce/NOAA/NMFS/NWFSC/Molecular Biology Protocols

Diatomaceous Earth-based Midi-prep

Note: This procedure is the method of choice for isolating double stranded plasmid-based templates for the Sequenase Dye-Labeled Terminator Sequencing Reactions.

A. Materials & Methods:

  1. Pick one colony into 3 ml of 2xTY containg the appropriate antibiotic (typically Amp at 25 ug/ml for pUC-based plasmids) and incubate at 37oC with shaking for 8-9 hours.

  2. Transfer seed culture to 50 ml media and incubate as above for 11-14 hours.

  3. Harvest cells by transferring into a 50 ml sterile conical tube and spinning at 3000 rpm in GPR tabletop centrifuge for 5 min. Decant supernatant and freeze cell pellet at -70oC for at least 1 hour.

  4. Resuspend defrosted cells in 2 ml GET/lysozyme (2 mg/ml). Add 4 ml of NaOH/SDS and incubate on ice for 5min.

  5. Add 4 ml of 3M NaOAc, pH4.8, and incubate on ice for 60min.

  6. Filter the supernatant through cheesecloth into another 50 ml conical tube and spin at 3000 rpm in the GPR centrifuge for 20min.

  7. Decant supernatant to 50 ml polypropylene tube and add 20 mg/ml of DNase-free RNaseA. Incubate at 37oC for 30 minutes.

  8. Add 7 ml (equal volume) of matrix (20mg/ml in guanadine HCl dissolved in 10:1 TE). Mix about 5min. Spin 5min at 3000 rpm in GPR tabletop centrifuge.

  9. Wash with an equal volume of wash buffer. Spin. Decant supernatant.

  10. Wash with an equal volume of acetone. Spin. Decant supernatant.

  11. Dry in vacuum oven (about 15-20min).

  12. Add 600 ul of 10:1 TE, incubate for 10min at 65oC with intermittent mixing.

  13. Spin at 8000 for 5min. Decant supernatant to Eppendorf tube.

  14. Spin again and transfer to Eppendorf tube (about 250 ul).

  15. Precipitate with 2.5 volumes of ethanol/acetate, rinse with 80% ethanol, and dry in a Speed-vac.

  16. Resuspend in 40 ul of 10:0.1 TE and assay concentration using 2 ul on an agarose gel. The yield should be ~1 ug/ml of starting cells. Use ~5 ug in the Sequenase Dye-Labeled Terminator Sequencing Reactions.

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