Preparation of DNA for Electroporation
I. Prepare DNA
- Inoculate LB culture, plus antibiotic, with a single colony.
- Grow overnight.
- Prepare DNA as per the Qiagen Maxi Prep Protocol.
- Quantify DNA with Spectrophotometer. [DNA] >1 mg/ ml is helpful for following steps.
II. Digest 300 ug plasmid DNA with appropriate enzyme
- 1 to 5 units enzyme / mg DNA.
- Digest 4 to 6 hours at appropriate temperature.
- Run a sample of cut DNA against uncut DNA on a gel to check for linearization.
- Add EDTA to 50 mM to stop reaction. Store at -20°C if necessary.
- Add TE to 600 ul.
- Phenol/CHCl3 extract twice.
- CHCl3 extract once.
- Add 1/10 volume 3M NaOAc.
- Add 2X volume 100% EtOH.
- Store at -20°C if necessary.
IV. Preparation of DNA the Day of Electroporation
- Spin down DNA, decant supe.
- Wash Pellet in 70% EtOH.
- Fill tube with 100% EtOH and take to tissue culture lab.