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Plasmid DNA Miniprep: Alkaline Lysis Method Options: (1) For 10 mls of culture: double volumes in steps 3 5; use 0.6 volumes (0.5 ml) of
 

Summary: Plasmid DNA Miniprep: Alkaline Lysis Method
Options: (1) For 10 mls of culture: double volumes in steps 3 5; use 0.6 volumes (0.5 ml) of
isopropanol in step 7; resuspend in 100 l TE at step 10. (2) Phenol:CHCl3 extract before step 7.
1. Inoculate 5 ml of LB + antibiotic in 15 ml round bottom tube with a single bacterial colony. Or,
inoculate 1.5 ml of TYGPN + antibiotic in 2.0 ml tube. Grow to saturation at 30 37C.
2. Place on ice. Pellet cells (10 min at 2000 rpm, 1 min in microfuge). Discard supernatant.
3. Resuspend pellet in 100 l GTE or TE buffer. If in 15 ml tube, transfer to 1.5 ml microfuge tube.
4. Add 200 l 0.2 N NaOH/1% SDS solution. Mix completely by inverting and shaking tube. Do not
vortex. Place on ice for 5 minutes.
NaOH/SDS solution: freshly dilute 2 M NaOH and 10% SDS each at 1 in 10.
5. Add 150 l of 3 M potassium acetate, pH 5. Immediately vortex for exactly 5 seconds to mix.
Place on ice for 5 minutes.
6. Spin 3 minutes at maximum speed in microfuge to pellet cell debris and chromosomal DNA.
7. Transfer supernatant to fresh 1.5 ml tube. Add 2 volumes (0.8 ml) of 100 % ethanol. Vortex to
mix well. Hold at room temperature 1 minute to precipitate nucleic acids.
8. Spin 1 minute at room temperature in microfuge to pellet nucleic acids. Discard supernatant.
9. Add 0.5 ml of 70% EtOH. Dislodge pellet (scrape across microfuge tube rack). Spin 1 minute.
Discard supernatant. Spin again for a few seconds. Discard remaining 70% EtOH.
10. Resuspend pellet in 50 l TE, pH 8.0 + 10 g/ml RNase A. Hold at room temperature for 1 hour.
RNase A dilute 1 l 10 mg/ml RNase A into 1 ml TE. Use 1-2 l for restriction digest.

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine