Wittrup: Plasmid miniprep

From OpenWetWare

Revision as of 17:45, 6 March 2006 by Lippow (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

Materials

QIAprep Spin Miniprep Kit (Qiagen 27106)

E. coli culture

Protocol

This protocol is adapted/condensed from the Qiagen instructions. The Qiagen site ([1]) offers several helpful tips that are useful for first-time users and for troubleshooting.

1. Grow 1-5 mL of E. coli for 12-16 h at 37ºC.

2. Pellet culture and remove supernatant (3 min. at >6800g is sufficient).

3. Add 250 μL of resuspension Buffer P1 to pelleted cells in microcentrifuge tube and resuspend cells.

4. Add 250 μL of lysis Buffer P2 to tube. Invert several times until solution is viscous and homogeneously blue (or slightly clear if no LyseBlue was added).

5. Add 350 μL of neutralizing Buffer N3 to tube. Invert several times until suspension is clear and clumps form.

6. Centrifuge at 15,000-18,000g for 10 min.

7. Add supernatant to a QIAprep column in a collection tube by pouring or pipetting.

8. Centrifuge the column at 15,000-18,000g for 30-60 s and discard flowthrough.

Note: If the strain has high carbohydrate content or high nuclease activity (such as an endA+ strain) wash with 500 μL of Buffer PB, centrifuge, and discard flowthrough.

9. Add 750 μL of wash Buffer PE to column,

10. Centrifuge the column at 15,000-18,000g for 30-60 s and discard flowthrough.

11. Centrifuge the column at 15,000-18,000g for 60 s and discard flowthrough (to eliminate residual ethanol).

12. Place QIAprep column in 1.5 mL tube.

13. Add 50 μL of elution Buffer EB to the center of the column. Let stand for 60 s.

14. Centrifuge at 15,000-18,000g for 60 s.

15. Store DNA collected in vial at -20˚C.

Personal tools