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Houston, Texas

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Mouse Embryonic Stem Cell Core Facility
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Preparation of DNA for Electroporation

I. Prepare DNA

  1. Inoculate LB culture, plus antibiotic, with a single colony.
  2. Grow overnight.
  3. Prepare DNA as per the Qiagen Maxi Prep Protocol.
  4. Quantify DNA with Spectrophotometer. [DNA] >1 mg/ ml is helpful for following steps.

II. Digest 300 ug plasmid DNA with appropriate enzyme

  1. 1 to 5 units enzyme / mg DNA.
  2. Digest 4 to 6 hours at appropriate temperature.
  3. Run a sample of cut DNA against uncut DNA on a gel to check for linearization.
  4. Add EDTA to 50 mM to stop reaction. Store at -20°C if necessary.

III. Purification

  1. Add TE to 600 ul.
  2. Phenol/CHCl3 extract twice.
  3. CHCl3 extract once.
  4. Add 1/10 volume 3M NaOAc.
  5. Add 2X volume 100% EtOH.
  6. Store at -20°C if necessary.

IV. Preparation of DNA the Day of Electroporation

  1. Spin down DNA, decant supe.
  2. Wash Pellet in 70% EtOH.
  3. Fill tube with 100% EtOH and take to tissue culture lab.