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Blunt End Ligation - Revision history
2026-10-18T05:36:27Z
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Paul.xu at 13:45, 5 July 2017
2017-07-05T13:45:20Z
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<p><b>New page</b></p><div>== PCR ==<br />
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1, Run PCR using Pfu or Phusion DNA Polymerase. Do not use Taq DNA polymerase<br />
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2, Run PCR product on agarose gel.<br />
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3. Cut out desired band, add gel solubilization buffer QG.<br />
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== Vector Digestion ==<br />
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4. Digest pET30b:<br />
<blockquote>5 µl of pET30b<br/>0.5 µl of EcoRV<br/>0.5 µl of CIP<br/>1.5 µl of 10X NEB Buffer 3<br/>1.5 µl of 10X BSA<br/>water to 15 µl.<br/>37 oC for 1 hour.<br/></blockquote><br />
5. Run gel and cut out linearilzed vector.<br />
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6. Add some QG buffer, dissolve gel with QG buffer, mix with solution from step 3.<br />
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7. Purify DNA using spin column.<br />
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8. Elute DNA using 20 µl water.<br />
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9. Add 2.5 µl 10X ligase buffer.<br />
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10. Add 0.5 µl T4 ligase.<br />
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11. Ligate at room temperature for 2 hours.<br />
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12. Transform 100 µl Top10 competent cells with 10 µl ligation mix.<br />
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Ligase buffer:<br />
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Contains 5% PEG<br />
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10x Ligation buffer (containing 1 ul of 10 mM ATP) 2.5 µl T4 DNA ligase 0.5 µl 30% PEG 8000 4 µl<br />
[[Category:Protocols]]<br />
10X Ligase buffer: 0.5 M Tris pH 7.8, 0.1 M MgCl2, &nbsp;50 mM DTT, 2 mM EDTA, &nbsp;4 mM rATP, 5% PEG 8000<br />
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Vector: add&nbsp;1 µl Biolabs calf intestine alkaline phosphatase. Incubate 37° for 1 hr.</div>
Paul.xu