LARGE SCALE PLASMID PREP (QIAGEN MAXIPREP)

1. Grow fresh colony in 100 ml LB/AMP

2. Harvest cells by centrifugation 6000 x g 15 minute at 4 degrees.

3. Resuspend pellet in 10 ml buffer P1

4. Transfer solution to a 30 ml centrifuge tube.

5. Add 10 ml buffer P2, invert to mix, set at room temp for 5 minutes.

6. Add 10 ml of cold buffer P3, mix gently, set on ice for 20 minutes.

7. Centrifuge > 20K x g for 30 minutes.

8. Immediately transfer supernatant to clean 30 ml cent. tube and repeat spin (20K x g) for 15 minutes.

9. While spinning, equilibrate a Quiagen tip 500 with 10 ml of buffer QBT.

10. Promptly apply supernatant from step 8 to column.

11. Wash column twice with 15 - 30 ml of buffer QC.

12. Elute DNA with 15 ml of buffer QF.

13. Precipitate DNA with 10.5 ml of room temp isopropanol.

14. Centrifuge 15K x g for 30 minutes at 4 degrees.

15. Wash the DNA pellet with 70 % ethanol and centrifuge 15 K x g for 15 minutes.

16. Speed dry pellet in the speed vac (~10 minutes).

17. Resuspend the pellet in 200ul of ddWater.