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1037 p8H-T7ligase - Revision history
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Paul.xu at 19:30, 11 June 2012
2012-06-11T19:30:21Z
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<p><b>New page</b></p><div>== '''Cloning of 1037 p8H-T7ligase into&nbsp;p8H-T7ligase''' ==<br />
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'''Purpose:''' Cloning of 1037 p8H-T7ligase 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: 1037''' <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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6/5/2012: No growth on plates, redid from digestion <br />
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[[Image:1037 1143 Digestion 1197 1154 PCR Product.JPG]] <br />
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Digested with&nbsp;Nde1 + Xho1, and cut of vector&nbsp;p8H (1135), purified, eluded with 20 uL water, ligated and transformed with DH5 alpha cells, grew on KAN plate over night <br> <br />
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06/06/2012: No growth on plates, redid PCR&nbsp;product, no product, do experiment again, maybe synthesized template incorrectly or PHUSION&nbsp;enzyme not efficient? <br />
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[[Image:1036 Amp.JPG]] <br />
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Redid amplication, cut out band, purified it for digestion <br />
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[[Image:1037 amp 2.JPG]] <br />
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Digested with Nde1 + Xho1, and cut of vector p8H (1135), purified, eluded with 20 uL water, ligated and transformed with DH5 alpha cells, grew on KAN plate 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:1037 0607 AMP.JPG]] <br />
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Digested with Nde1 + Xho1, and cut of vector p8H (1135), purified, eluded with 20 uL water, ligated and transformed with DH5 alpha cells, grew on KAN plate over night <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;06082012<br> <br />
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[[Image:0608 PCR Screening.JPG]] <br />
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All samples (1-7)&nbsp;were positive, grew samples 1-6 in KAN&nbsp;media overnight (lanes 12-17) <br />
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[[Image:1037 0611 Digestion.JPG]] <br />
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Prepared MiniPrep on samples, digested with enzymes to check for inserts, submitted sample 3 (lane 9)&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