Editing 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.

&nbsp;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&nbsp;50~100 ng/ul&nbsp; for DNA sequencing.

[[File:CATB Ca Mg on Isopropanol Plasmid4.JPG|RTENOTITLE]][[File:CATB Ca Mg on Isopropanol Plasmid2.JPG|RTENOTITLE]]

[[File:CATB Ca Mg on Isopropanol Plasmid3.JPG|RTENOTITLE]][[File:CATB Ca Mg on Isopropanol Plasmid.JPG|RTENOTITLE]]

[[File:Isopropanol-PEG-Plasmid QC digestion.JPG|RTENOTITLE]][[File:Plasmid isopropanol ppt.JPG|RTENOTITLE]][[File:PEG ppt Plasmid.JPG|RTENOTITLE]]

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.

[[File:Recover DNAfrom gel.png|RTENOTITLE]]



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&nbsp; (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
[[Category:Protocols]]