https://eln.excellgen.com/index.php?action=history&feed=atom&title=Plasmid_DNA_purification_using_isopropanol_and_PEGPlasmid DNA purification using isopropanol and PEG - Revision history2026-10-18T08:48:35ZRevision history for this page on the wikiMediaWiki 1.44.0https://eln.excellgen.com/index.php?title=Plasmid_DNA_purification_using_isopropanol_and_PEG&diff=20095&oldid=prevPaul.xu at 13:39, 12 August 20192019-08-12T13:39:45Z<p></p>
<p><b>New page</b></p><div>1. Spin down 2~5 ml cells.<br />
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2. Resuspend in 0.25 ml P1<br />
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3. Add 0.25 ml P2, mix by inverting.<br />
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4. Digest RNA for 2 ~5 min<br />
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5. Add 0.35 ml N3.<br />
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6. Spin for 2X 2.5 min, reposition tube between spins<br />
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7. Transfer supernatant to another clean tube.<br />
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8. Add 0.65 ml isopropanol<br />
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9. Spin 5 min.<br />
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10. Dump off liquid.<br />
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11. Add 100 ~200 ul 70% ethanol<br />
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12. Spin 15~30 sec.<br />
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13. Dump off ethanol<br />
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14. Spin 5 sec.<br />
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15. Remove liquid using pipette.<br />
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16. Add 50 ul EB elution buffer.<br />
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&nbsp;17. Votex.<br />
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18. Use 3~5 ul for digestion<br />
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19. Spin 1 min, carefully transfer liquid to another clean tube.<br />
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20. Add 50 ul of 2 X PEG/NaCl solution (20% PEG8000, 1.6 M NaCl). This step remove RNA<br />
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21. Spin 5 min<br />
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22. Dump off liquid.<br />
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23. Add 100 ~200 ul 70% ethanol<br />
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24. Spin 15~30 sec.<br />
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25. Dump off ethanol<br />
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26. Spin 5 sec.<br />
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27. Remove liquid using pipette.<br />
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28. Add 50 ul EB elution buffer.<br />
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29. Measure DNA using nanodrop.<br />
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30. Dilute to&nbsp;50~100 ng/ul&nbsp; for DNA sequencing.<br />
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[[File:CATB Ca Mg on Isopropanol Plasmid4.JPG|RTENOTITLE]][[File:CATB Ca Mg on Isopropanol Plasmid2.JPG|RTENOTITLE]]<br />
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[[File:CATB Ca Mg on Isopropanol Plasmid3.JPG|RTENOTITLE]][[File:CATB Ca Mg on Isopropanol Plasmid.JPG|RTENOTITLE]]<br />
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[[File:Isopropanol-PEG-Plasmid QC digestion.JPG|RTENOTITLE]][[File:Plasmid isopropanol ppt.JPG|RTENOTITLE]][[File:PEG ppt Plasmid.JPG|RTENOTITLE]]<br />
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If spin column yield is low, need to regenerate column with 100 ul of 1 N NaOH for 5 min. Spin and discard NaOH.<br />
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Looks like column recovery is higher than isopropanol.<br />
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[[File:Recover DNAfrom gel.png|RTENOTITLE]]<br />
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If Miniprep column is too old (binding affinity becomes too weak), add 100 ul 1 N NaOH to soak the membrane for 5 min.<br />
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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.<br />
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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<br />
[[Category:Protocols]]</div>Paul.xu