Miniprep Plasmid Extraction Protocol

1. Resuspend cell pellet in 250 l of P1. 2. Add 250 l of P2 and invert several times to lyse cell membranes. 3. Add 350 l of N3 and invert several times to neutralize the lysis reaction. 4. Centrifuge at 17 Kg for 10 minutes. 5. Apply supernatant to spin column. 6. Centrifuge for 30 seconds at max speed and discard flowthrough. 7. Wash with 500 l of PB, centrifuge for 30 seconds, and discard the flowthrough (this step is optional, for neutralizing high-nuclease activity cell lines. Do not use for DH5-alpha cells). 8. Wash with 750 l of PE, centrifuge for 1 minute, discard the flowthrough, and centrifuge again for 30 seconds. 9. Place the column into a new eppendorf, add 50 l of EB or Milli-Qgrade water to elute the DNA. Let sit for 1 minute, and centrifuge for 1 minute. 10. Store purified DNA at -20oC or lower. Note: All centrifugation steps are done at the maximum speed.

Appendix: Recipes for Qiagen Miniprep buffers. P1 50 mM Tris-HCl pH 8.0 10 mM EDTA 100 g/ml RNaseA PB 5 M Gu-HCl 30% isopropanol P2 200 mM NaOH 1% SDS Optional pH indicator is thymophthalein PE 10 mM Tris-HCl pH 7.5 80% ethanol N3 4.2 M Gu-HCl 0.9 M potassium acetate pH 4.8 EB 10 mM Tris-Cl, pH 8.5