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1155 pET28b-Ara-S1 - Revision history
2026-10-18T02:17:33Z
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Paul.xu at 18:31, 11 June 2012
2012-06-11T18:31:40Z
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<p><b>New page</b></p><div>== '''Cloning of Arabidopsis S1 Nuclease into&nbsp;1155 pET28b''' ==<br />
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'''Purpose:''' Cloning of&nbsp;Arabidopsis S1 Nuclease into&nbsp;1155 pET28b <br />
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'''Date:&nbsp;''' 06/05/2012<br> <br />
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'''Notebook:&nbsp;''' <br />
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'''Page: 008803-003308''' <br />
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'''Clone ID: 1155''' <br />
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'''Experimenters:'''&nbsp; Jennifer Truong<br> <br />
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== '''Cloning''' ==<br />
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See [http://www.excellgen.net/eln/index.php/1154_pCAMBIA-2301-Ara-s1 1154]&nbsp;(mistake of name, using a different plasmid) <br />
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6/5/2012: 1154-5 sequencing came back mutated, restarted experiment from the beginning: <br />
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[[Image:1155 Digestion.JPG]] <br />
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Synthesized PCR product with PHUSION enzyme, ran PCR gel, cut out product for purifaction <br />
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[[Image:1155 PCR Product.JPG]] <br />
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Digested 1155 and pET28b (vector) with Nco1 + Xho1, cut out bands, ligated and transformed cells with DH5 alpha and grew on KAN plates<br> <br />
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06/06/12: Nothing grew on the KAN&nbsp;plates <br />
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Cut out the wrong band from the day before (see above), redid gene synthesis and cut out circled band[[Image:1155 Amp.JPG]] <br />
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Digested purified DNA and pET28b (V12)&nbsp;vector with Nco1 +&nbsp;Xho1, cut out bands for ligation <br />
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[[Image:1155 Digestion 06062012.JPG]] <br />
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Redid gene synthesis, cut out band, purified for digestion <br />
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[[Image:1155 amp 2.JPG]] <br />
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Digested purified DNA and pET28b (V12) vector with Nco1 + Xho1, cut out bands for ligation <br> <br />
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[[Image:06062012 Digestion.JPG]] <br />
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06/07/2012: Again, no growth on plates. <br />
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Synthesized PCR&nbsp;product again, cut out circled band, purified DNA, digested DNA and vector for digestion <br />
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[[Image:1155 0607 AMP.JPG]] <br />
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Digested purified DNA and pET28b (V12) vector with Nco1 + Xho1, cut out bands for ligation (lanes 1 and 2)<br><br />
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[[Image:0607_Digestion.JPG]]<br />
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== '''Colony Screening'''<br> ==<br />
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Date:&nbsp;06/08/2012<br> <br />
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[[Image:0608 PCR Screening.JPG]] <br />
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All screening were positive, grew samples 1-6 in media (lanes 1-6) <br />
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[[Image:1197_1155_Digestion.JPG]]<br />
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Performed miniprep on samples, digested with enzymes to check for inserts, submitted sample #2 (lane 8)&nbsp;for sequencing.<br />
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== '''Result intepretation/Summary''' ==<br />
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== '''Conclusion''' ==<br />
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[[Category:Cloning]]</div>
Paul.xu