Plasmid Mini-Prep from E. coli

P1 (store @ 4 deg)

12.5 ml 1M Tris, pH 8

5 ml 0.5M EDTA

232.5 ml H₂O

P2 (store @ RT, make fresh after 2-4 weeks!)

25 ml 10% SDS

50 ml 1N NaOH

175 ml H₂O

P3 (store @ 4 deg)

3 M KOAc, pH 5.5

RNase A

Dissolve powder in H₂O to give 10 mg/ml

Boil 5 min to kill DNase

Aliquot and store at –20 deg.

1. Setup an overnight culture of 2 ml.

2. Spin down 1-1.5 ml 10 sec pulse-spin.

3. Aspirate medium.

4. Suspend cells in 100 ul P1 by vortexing.

5. Add 100 ul P2 and mix by inversion

5 min @ RT

6. Add 100 ul cold P3, mix by tapping.

5 min on ICE

7. Spin 1 min.

8. Take 250 ul of supe and add to 5 ul RNase A (10 mg/ml) in a fresh tube.

20 min @ 37 deg

9. Add 200 ul phenol and 200 ul CH₃Cl/Isoamyl Alcohol, vortex and spin for 1 min.

10. Take 200 ul of aqueous (upper) phase and add to 500 ul cold 100% EtOH.

11. Sin for 20-30 min. (This can be RT or cold spin.)

12. Wash with 70% EtOH.

13. Dry in Speed Vac.

14. Resuspend in 25 ul H₂O.

15. Cut 1 ul to check.

Notes:

§ This prep works great for sequencing unless there is RNA contamination. If RNA contamination is still present and undesired, add 1 ul RNase A (10 mg/ml). If you’re going to sequence, then you’ll need to remove the RNase A by phenol extraction or qiaquick column.