https://eln.excellgen.com/index.php?action=history&feed=atom&title=549_pET28b-Phi29DNAPol 2026-10-18T08:18:17Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=549_pET28b-Phi29DNAPol&diff=7732&oldid=prev 2013-01-02T18:35:07Z

<p></p> <p><b>New page</b></p><div>4/8/2012 <br /> <br /> Gene synthesis of 549 with Kapa Hi Fi, 25 cycles <br /> <br /> Fragment 1: using oligo mix 1-1 to 1-26, using 1-1 &amp;amp; 1-26 primers <br /> <br /> Fragment 2: using oligo mix 2-1 to 2-27, using 2-1 &amp;amp; 2-27 primers <br /> <br /> [[Image:203 549 4 8 2012.jpg]] <br /> <br /> 4/8/2012: Fragment 2 gene synthesis <br /> <br /> [[Image:Phi29 gene synthesis 4 8 2012.jpg]] <br /> <br /> cut out band and reamplify. <br /> <br /> [[Image:Phi29 gene synthesis 4 9 2012.jpg]] <br /> <br /> 4/10/2012&lt;br&gt; <br /> <br /> Gibson Assembly of fragment 1 + 2 and vector. Use 5 ul mixture with enzyme mix (Gibson or SystemBio)&lt;br&gt; <br /> <br /> [[Image:Phi29 Gibson assembly 4 10 2012.jpg]]&lt;br&gt; <br /> <br /> Lane 1: fragment 1 + Fragment 2 + pET28b, no Gibson mix is added&lt;br&gt; <br /> <br /> Lane 2:&amp;nbsp; fragment 1 + Fragment 2 + pET28b, use systembio reagent&lt;br&gt; <br /> <br /> Lane 3: fragment 1 + Fragment 2 + pET28b, add 15 ul Gibson Mix.&lt;br&gt; <br /> <br /> Cut out bands, gel purify and transform Top10. <br /> <br /> [[Image:203 turboTEV gene syn 4 11 2012.jpg]] <br /> <br /> == &#039;&#039;&#039;Cloning of 549_pET28b-Phi29DNAPol&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of 549_pET28b-Phi29DNAPol <br /> <br /> &#039;&#039;&#039;Date:Dec&#039;&#039;&#039;, 2012&lt;br&gt; <br /> <br /> &#039;&#039;&#039;Notebook:&amp;nbsp;&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Page:&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Clone ID: &#039;&#039;&#039;549 <br /> <br /> &#039;&#039;&#039;Experimenters: &#039;&#039;&#039; shanyi <br /> <br /> == &#039;&#039;&#039;Results&#039;&#039;&#039; ==<br /> <br /> === Cloning ===<br /> <br /> gene synthesis 2 fragments.&lt;br&gt; <br /> <br /> [[Image:549-1 549-2 1444 1478 1517 1480pcr.JPG]] <br /> <br /> join 2 fragments by PCR&lt;br&gt; <br /> <br /> [[Image:1279 1118 1514 1464 1395 1279 1143DIGEST.JPG]]&lt;br&gt; <br /> <br /> digest pET28b WITH Nco1+ Xho1&lt;br&gt; <br /> &lt;pre /&gt; <br /> &lt;br&gt; <br /> <br /> === &lt;u&gt;&lt;/u&gt;Colony Screening&lt;br&gt; ===<br /> <br /> [[Image:549digest.JPG]] <br /> <br /> == &#039;&#039;&#039;Result intepretation/Summary&#039;&#039;&#039; ==<br /> <br /> &lt;br&gt; <br /> <br /> == &#039;&#039;&#039;Conclusion&#039;&#039;&#039; ==<br /> <br /> &lt;br&gt; <br /> <br /> === Cloning ===<br /> <br /> [[Category:Cloning]]</div>

Paul.xu