| | |
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 µl 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 37°C.
2. Place on ice. Pellet cells (10 min at 2000 rpm, 1 min in microfuge). Discard supernatant.
3. Resuspend pellet in 100 µl GTE or TE buffer. If in 15 ml tube, transfer to 1.5 ml microfuge tube.
4. Add 200 µl 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 µl 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 µl TE, pH 8.0 + 10 µg/ml RNase A. Hold at room temperature for 1 hour.
RNase A dilute 1 µl 10 mg/ml RNase A into 1 ml TE. Use 1-2 µl for restriction digest.
|
