https://eln.excellgen.com/index.php?action=history&feed=atom&title=Cloning_after_PCRCloning after PCR - Revision history2026-10-18T08:44:08ZRevision history for this page on the wikiMediaWiki 1.44.0https://eln.excellgen.com/index.php?title=Cloning_after_PCR&diff=17408&oldid=prevCrm at 16:47, 21 March 20182018-03-21T16:47:42Z<p></p>
<p><b>New page</b></p><div>'''Insert:'''<br />
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1, PCR amplify a desired fragment from either miniprep DNA, genomic DNA, cDNA from mRNA, or gene synthesis<br />
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2. Run gel to isolate desired fragment<br />
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3, Cut out band<br />
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4, Add 500~800 ul QG Buffer<br />
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5, Dissolve gel at 50~60 oC<br />
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7. Purify using a mini spin column<br />
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8, elute with 25 ul H2O<br />
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9, Add 3 ul of 10X Buffer (see NEB chart for NEB buffer or use FastDigest buffer)<br />
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10, Add 0.5~ 1 ul restriction enzyme<br />
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11. Digest at 37 oC for 30 min to 60 min.<br />
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'''Vector'''<br />
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From step 9, digest ~150 ng of vector with the same enzymes or compatible enzymes for 30 min at 37 oC.<br />
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12. run gel cut out desired vector band<br />
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13, Add QG, dissolve<br />
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14, Add digested insert, from '''step 11''' mix.<br />
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15, Purify with a spin column<br />
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16, Elute with 25 ul H2O<br />
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'''Ligation'''<br />
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Add 3 ul 10 X T4 ligase buffer, 1 ul T4 DNA ligase<br />
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Ligate at RT &nbsp;for 30 min<br />
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'''Transformation'''<br />
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See [[Transformation|transformation protocol]]<br />
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== [[Blunt-end_PCR_Cloning|Blunt End Cloning]] ==<br />
[[Category:Protocols]]</div>Crm