Team:Wisconsin-Madison/Plasmid Preparation by Boiling Lysis

Plasmid Preparation by Boiling Lysis

Source

Hybrid of Maniatis and Qiagen

Materials

• Qiagen PCR purification kit
• Isopropanol
• Sodium Acetate (2.5 M, pH 5.2)
• STET

(10 mM Tris-HCl (pH 8.0), 0.1 M NaCl, 1 mM EDTA (pH 8.0), 5% (v/v) Triton X-100)

• Lysozyme (10mg/ml in 10 mM Tris-HCl, pH 8.0) Make Fresh each time

Protocol

1. Spin down cells (4-6 ml of overnight is fine)

2. Set up boiling water bath.

3. Prepare fresh lysozyme mix.

4. Aspirate cell pellet when centrifugation is complete.

5. Resuspend cells in 350 µl of STET, do so quickly by vortexing.

6. Make sure boiling bath is ready.

7. Add 25 ul of lysozyme to the resuspended pellet.

8. Set timer for 40 seconds.

9. Place cells in bath for exactly 40 seconds.

10. Centrifuge at max speed for 15 minutes at room temp.

11. Prepare fresh 2.0 ml tubes for each sample

12. Transfer the supernatant to a fresh tube.

13. Precipitate the nucleic acids by adding 40 µl of 2.5 M sodium acetate (pH 5.2), and 420 µl of isopropanol.

14. Mix by lightly vortexing, and let stand at room temp for 5 minutes.

15. Pellet the nucleic acids by centrifuging at maximum speed for 10 minutes at 4 oC.

16. Remove all isopropanol, by aspirating and then speed-vacing for 10 minutes.

17. Resuspend in 50-100 µl of Buffer P1 with RNase H to remove RNA.

18. Run PCR purification kit to clean up plasmid. Maniatis uses ethanol washes, and resuspension in RNase A containing TE.