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203-pET28bTurboTEV - Revision history
2026-10-18T13:48:44Z
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Xiaojing.Wang at 19:54, 13 April 2012
2012-04-13T19:54:48Z
<p></p>
<p><b>New page</b></p><div>This is done by Paul <br />
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TurboTEV gene synthesis using Kapa HiFi <br />
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4/7/2012: One step: <br />
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[[Image:1012 1078 gene synthesis 4 7 2012.JPG]]<br> <br />
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4/8/201: two steps <br />
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first reaction: <br />
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tube one: oligo 1-10, 0.25 ul (0.5 unit) Kapa <br />
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tube two: oligo 11-20, 0.25 ul (0.5 unit) kapa <br />
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25 ul final volumn without amplifying primers (outer primers). Amplify for 5 cycles. <br />
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while they are still hot (95oc), transfer tube 2 to tube 1, add 2 ul of outer primer mixture (F &amp; R). <br />
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Amplify 25 cycles. <br />
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[[Image:TurboTEV gene synthesis 4 8 2012.JPG]] <br />
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4/8/2012 <br />
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digest PCR product with EcoR1 + Sal1, digest pET28b with EcoR1 + Sal1 <br />
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cut out/purify bands and ligate <br />
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[[Image:203 549 4 8 2012.jpg]] <br />
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<br> <br />
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[[Image:203 turboTEV gene syn 4 11 2012.jpg]] <br />
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Select 6 clones to mini-prep<br>203 plasmids are digested by EcoR1 + Sal1 <br> <br />
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[[Image:203 digest 1 4.13.2012.JPG]]<br> <br />
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203 plasmids are digested by Xho1<br> <br />
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[[Image:203 digest 2 4.13.2012.JPG]] <br />
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203-1~203-6&nbsp; may be correct.<br> <br />
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Select 203-3 to sequence.<br />
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[[Category:Cloning]]</div>
Xiaojing.Wang