Plasmid DNA purification using isopropanol and PEG
1. Spin down 2~5 ml cells.
2. Resuspend in 0.25 ml P1
3. Add 0.25 ml P2, mix by inverting.
4. Digest RNA for 2 ~5 min
5. Add 0.35 ml N3.
6. Spin for 2X 2.5 min, reposition tube between spins
7. Transfer supernatant to another clean tube.
8. Add 0.65 ml isopropanol
9. Spin 5 min.
10. Dump off liquid.
11. Add 100 ~200 ul 70% ethanol
12. Spin 15~30 sec.
13. Dump off ethanol
14. Spin 5 sec.
15. Remove liquid using pipette.
16. Add 50 ul EB elution buffer.
17. Votex.
18. Use 3~5 ul for digestion
19. Spin 1 min, carefully transfer liquid to another clean tube.
20. Add 50 ul of 2 X PEG/NaCl solution (20% PEG8000, 1.6 M NaCl). This step remove RNA
21. Spin 5 min
22. Dump off liquid.
23. Add 100 ~200 ul 70% ethanol
24. Spin 15~30 sec.
25. Dump off ethanol
26. Spin 5 sec.
27. Remove liquid using pipette.
28. Add 50 ul EB elution buffer.
29. Measure DNA using nanodrop.
30. Dilute to 50~100 ng/ul for DNA sequencing.
If spin column yield is low, need to regenerate column with 100 ul of 1 N NaOH for 5 min. Spin and discard NaOH.
Looks like column recovery is higher than isopropanol.
If Miniprep column is too old (binding affinity becomes too weak), add 100 ul 1 N NaOH to soak the membrane for 5 min.
If 0.6 vol of isopropanol is added to the supernant (0.25 ml P1, 0.25 ml P2, 0.35 ml N3), lot of RNA will bind to column.
After eluting plasmid DNA with EB, add 1 vol of 2 X PEG solution (20% PEG, 1.6 M NaCl). Mix and Spin 5 min