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1143 p8H-T7-Exo-1 - Revision history
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Jennifer.truong: /* Colony Screening */
2012-06-11T18:00:25Z
<p><span class="autocomment">Colony Screening</span></p>
<p><b>New page</b></p><div>== '''Cloning of 1143 p8H-T7-Exo-1 into&nbsp;p8H-T7ligase''' ==<br />
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'''Purpose:''' Cloning of 1143 p8H-T7-Exo-1 into p8H-T7ligase <br />
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'''Date:&nbsp;''' 06/04/2012<br> <br />
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'''Notebook: '''Jennifer Truong <br />
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'''Page: 003347-003349''' <br />
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'''Clone ID: 1143''' <br />
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'''Experimenters:'''&nbsp; Jennifer Truong<br> <br />
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== '''Cloning''' ==<br />
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Date: 06/04/2012<br> <br />
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PCR Template: <br />
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[[Image:1143 1037 Template.JPG]]<br> <br />
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Isolated band for cloning<br> <br />
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Digested with Nde1 ad Xho1, using p8H (1135) template <br />
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[[Image:1143 1037 Digestion.JPG]] <br />
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Cut out bands for ligation, transformed DH5 alpha cells and grew on KAN R plates <br />
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6/05/12: Plates showed no growth, restart from digestion <br />
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Digested with&nbsp;Nde1 + Xho1 <br />
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[[Image:1143 Digestion.JPG]] <br />
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Cut out bands (1143 DNA and p8H 1135 vector), purified DNA, ligated, transformed with DH5 alpha cells and grew on KAN R plates over night <br />
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06/06/2012: No growth overnight. Redid gene synthesis, cut out bands for digestion <br />
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[[Image:1043 Amp.JPG]] <br />
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Digested purified DNA and p8H&nbsp;vector with Nde1 and Xho1, cut out bands for ligation <br />
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[[Image:1143 Digestion 06062012.JPG]] <br />
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Redid amplifaction, cut out band, purified it for digestion <br />
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[[Image:1143 amp 2.JPG]] <br />
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Cut out bands (1143 DNA and p8H 1135 vector), purified DNA, ligated, transformed with DH5 alpha cells and grew on KAN R plates over night <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 product again, cut out circled band, purified DNA, digested DNA and vector for digestion<br> <br />
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[[Image:1143 0607 AMP.JPG]] <br />
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Digested purified DNA and p8H vector with Nde1 and Xho1, cut out bands for ligation (lanes 3 and 4)<br> <br />
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[[Image:0607 Digestion.JPG]] <br />
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06/08/2012: Very few colonies, amplified again with KAPA&nbsp;enzyme (20 cycles) <br />
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Cut out band for purification and digestion <br />
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[[Image:1143 0608 amp.JPG]] <br />
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Digested purified DNA and p8H vector with Nde1 and Xho1, cut out bands for ligation<br><br />
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[[Image:1143_0608_Digestion.JPG]]<br />
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== '''Colony Screening'''<br> ==<br />
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[[Image:0608 PCR Screening.JPG]]<br> <br />
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Grew samples 1 and 2 in media (lanes 19 and 20) <br />
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Prepared MiniPrep of samples, digested with Nde1 and Xho1 to confirm inserts<br />
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[[Image:1143_1225_Digestion.JPG]]<br />
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Submitted sample 1 for sequencing<br />
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== '''Result intepretation/Summary''' ==<br />
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== '''Conclusion''' ==<br />
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[[Category:Cloning]]</div>
Jennifer.truong