https://eln.excellgen.com/index.php?action=history&feed=atom&title=LigationLigation - Revision history2026-10-18T15:01:48ZRevision history for this page on the wikiMediaWiki 1.44.0https://eln.excellgen.com/index.php?title=Ligation&diff=14661&oldid=prevPaul.xu at 20:02, 24 March 20162016-03-24T20:02:24Z<p></p>
<p><b>New page</b></p><div>1. Digest plasmid or purified PCR product with 0.3~0.5 ul Fermentas or NEB enzymes with Fermentas FastDigest Buffer (green) or NEB Buffer.<br />
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Plasmid (e.g., vector): 37 oC for 30min to 1 hour. <br />
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Purified PCR product: 37 oC for 1~2 hours, sometimes overnight<br />
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2. Run agarose gel to purify vector and insert.<br />
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3. Cut out vector and PCR bands<br />
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4. Dissolve gel in QG gel-solubilization buffer at 55 ~65 oC. Mix insert (or digested PCR product) with vector.<br />
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5. Purify DNA using spin column: load both dissolved DNA solutions to a column, spin for 30 sec, then wash 2X 350 ul of PE (spin for 15 sec at max speed), then spin 1 min at mix speed.<br />
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6. Elute DNA using 20 ul autoclaved ddH2O.<br />
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7. Add 2.5 ul ligation buffer and 1 ul ligase.<br />
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8. Ligate at RT for 30 min to 2 hours, or overnight.<br />
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9. Use entire ligation reaction for transformation.<br />
[[Category:Protocols]]</div>Paul.xu