Kit-free Alkaline Lysis Plasmid Miniprep

Step 1. Grow 2 mL overnight cultures from single colonies of bacteria containing your plasmid of interest.

Step 2. Add 1.5 mL of the stock culture to a 1.75 mL microfuge tube.

Step 3. Centrifuge in microfuge tube at 10,000 g for 30 sec.

30 sec @ 10000 𝘨

Step 4. Pour off the supernatant, being careful not to disturb the bacterial pellet.

Step 5. Resuspend the pellet in 100 μL of cold Solution I.

Step 6. Vortex the solution for until all bacteria are fully resuspended.

Step 7. Add 200 μL of Solution II and invert the tube carefully 5 times to mix the contents. The contents will become clear and thicker as the proteins and DNA are denatured.

Do not vortex at this stage or the genomic DNA will become sheared and will therefore contaminate your purified plasmid DNA.

Step 8. Incubate solution on ice for 5 min.

5 min @ -20 °C

Step 9. Add 150 μL of cold Solution III to each tube.

Step 10. Mix by inverting several times. A white precipitate will be formed which contains the bacterial proteins and genomic DNA.

Step 11. Incubate tube on ice for 5 min.

5 min @ -20 °C

Step 12. Centrifuge the tube for 5 min at 12,000 g.

5 min @ 12000 𝘨

Pellet contains proteins, cell fragments, salt and other extra particles from solutions. Supernatant contains the plasmid DNA separated from bacterial chromosomes.

Step 13. Collect the supernatant into a new tube by pipetting or carefully pouring.

Step 14. Add 350 μL cold isopropanol to the solution to precipitate the plasmid DNA;

Step 15. Incubate the tube at -20 °C for 60 minutes or overnight.

60 min @ -20 °C

Step 16. Centrifuge solution at 16,000 rpm) for 30 minutes.

30 min @ 16000 𝘨

Step 17. Pour off supernatant tsaking care not to dislodge the pellet.

Step 18. Add 500 ul cold 70% ethanol.

This step removes excess salt from the pellet which may co-precipitate with DNA in isopropanol and can cause problems with some common reactions.

Step 19. Centrifuge solution at high speed (at least 12,000 rpm) for 20 minutes.

Step 20. Pour off supernatant tsaking care not to dislodge the pellet.

Step 21. Air dry the pellet (can be done by inverting the tube at an angle over kimwipe) for 30 minutes.

30 min

Step 22. Resuspend pellet with 25-50 μL of sterile H₂O.