https://eln.excellgen.com/index.php?action=history&feed=atom&title=Endotoxin-Free_Plasmid_from_Spheroplasts 2026-10-18T16:37:54Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=Endotoxin-Free_Plasmid_from_Spheroplasts&diff=10369&oldid=prev 2013-10-03T18:49:45Z

<p></p> <p><b>New page</b></p><div>1. Spin down cells from 1 L LB culture for 10 min at 16,000 X g&lt;br&gt;2. Resuspend pellet in 50 ml of cold 20% sucrose , 50 mM Tris-HCl, pH 8.0 . &lt;br&gt;3. Add 10 ml of freshly prepared lysozyme (5 mg/ml in 0.25 M Tris, pH 8.0) &lt;br&gt;4. Add 20 ml of cold 0.25M EDTA, pH 8.&lt;br&gt;5. Leave on ice for 5 min. &lt;br&gt;6. Add 20 ml of cold 0.25M Tris-HCl, pH 8.0, incubate at 37 oC for&amp;nbsp; 15 to 30 min.&lt;br&gt;7. Spin at 4000 g for 10 min, remove viscous supernatant carefully.&lt;br&gt;8. Resuspend spheroplast pellet in 30 ml of 50 mM glucose/25 mM Tris-HCl, pH 8.0/10 mM EDTA, pH 8.0 containing 100 ug/ml RNase A, or P1 containing purified RNAse or heat-treated RNase.&lt;br&gt;9. Incubate at 37 oC for 30 min or room temperature o/n.&lt;br&gt;10. Optional: Add Proteinase K to 100 ug/ml and incubate at 37 oC for 15 min.&lt;br&gt;11. Add 30 ml of P2. (0.2 M NaOH/1% SDS).&lt;br&gt;12. Add 30 ml of P3 (5 M KoAc, 11.5% glacial acetic acid) for anion exchange column, or N3 for silica membrane&lt;br&gt;13. Spin, filter supernatant using coffer paper.&lt;br&gt;14. Load to plasmid DNA purification column.&lt;br&gt; <br /> <br /> [[Category:Protocols]]</div>

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