https://eln.excellgen.com/index.php?action=history&feed=atom&title=SubCloningSubCloning - Revision history2026-10-18T05:22:06ZRevision history for this page on the wikiMediaWiki 1.44.0https://eln.excellgen.com/index.php?title=SubCloning&diff=20984&oldid=prevPaul.xu at 14:49, 17 June 20202020-06-17T14:49:37Z<p></p>
<p><b>New page</b></p><div>Digest Vector and Parent plasmid DNA at 1:3 ratio with restriction enzymes<br />
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use 15 ul reaction<br />
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for 30 min at 37 oC<br />
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Run on agarose gel. Must use fresh 0.5 X TAE buffer. Make sure wash gel tank with ddH2O<br />
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Cut out insert and vector.<br />
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Put insert and vector gel slice into the same 1.5 ml tube.<br />
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Add 1 ml QG buffer<br />
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incubate at 55 oC for 10 until gel slices are completely dissolved<br />
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Transfer liquid to a column, Spin for 30 s<br />
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Wash with 2 X 0.35 ml of PE buffer, spin 30 sec<br />
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Spin for another min at max speed<br />
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Add 25&nbsp;ul ddH2O, spin for 1 min&nbsp;at max &nbsp;speed.<br />
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Add 2.5 ul 10 X ligation buffer (must completely thawed and mixed by vortexing)<br />
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Add 1 ul T4 ligase<br />
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Ligate at room temperature for 1 hour<br />
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Add 100 ul NEB Turbo or DH5alpha<br />
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Incubate on ice for 20 min<br />
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Heat shock for 60 sec at 42 oC<br />
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Incubate on ice for 5 min<br />
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Add 1 ml SOC<br />
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Culture for 30 min to 1 hour at 37 oC in a shaker-incubator.<br />
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Spin down cells, dump liquid, leave only ~100 ul medium<br />
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Suspend cells and Spread on LB plate with antibiotics.<br />
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Grow at 37 oC o/n<br />
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Colony screening and restriction digestion, and sequencing if required.<br />
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Document all experiment details on ELN<br />
[[Category:Protocols]]</div>Paul.xu