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1137 p8H-Exo-VIII - Revision history
2026-10-18T07:29:31Z
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Paul.xu at 16:18, 23 July 2012
2012-07-23T16:18:35Z
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<p><b>New page</b></p><div>== '''Cloning of 1137 p8H-Exo-VIII''' ==<br />
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'''Purpose:''' Cloning of 1137 p8H-Exo-VIII <br />
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'''Date: '''June 26, 2012<br> <br />
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'''Notebook:&nbsp;''' <br />
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'''Page:''' <br />
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'''Clone ID: 1137''' <br />
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'''Experimenters: '''Jennifer Truong <br />
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== '''Results''' ==<br />
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=== Cloning<br> ===<br />
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Date: June 26, 2012<br> <br />
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[[Image:1137 amp.JPG]]<br> <br />
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Amplified DNA with PHUSION enzyme (0.5 uL , 25 cycles, 55 degees annealing temperature) <br />
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Cut out band (expected size = 2602 bp), purified and digested wth vector 1135 (p8H)&nbsp;with restriction enzymes Nde1 and Xho1<br> <br />
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[[Image:1137 digestion.JPG]] <br />
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Cut out bands in lanes 1 and 2, purified and ligated together overnight. <br />
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Date:&nbsp;July 18, 2012<br> <br />
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[[Image:1137 0718 Amp.JPG]]<br> <br />
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Cut out bands in lane 1 and 2 (expected size = 2602 bp), purified and digested wth vector 1135 (p8H) with restriction enzymes Nde1 and Xho1<br> <br />
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[[Image:1137_0718_digestion.JPG]]<br><br />
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Cut out bands in lanes 1 (insert)&nbsp;and 2 (vector), purified and ligated together overnight. <br><br />
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=== Colony Screening ===<br />
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[[Image:1137 Screening.JPG]]<br> <br />
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Screened coloines, samples 3, 4, 5, 6, 7 (?) and 8 were positive. <br />
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Grew in media overnight, do MiniPrep tomorrow <br />
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Date: June 29, 2012 <br />
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[[Image:0629 Screening.JPG]] <br />
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Digested Mini prep (see lanes 1-6)with Nde1 + Xho1<br> <br />
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Sent sample 3 (lane 3) to sequencing. <br />
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Date:&nbsp;July 12, 2012<br> <br />
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[[Image:0712 Screening.JPG]]<br> <br />
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Sequencing came back bad, grow more bacteria from stock solution, redid MiniPrep, digested plasmids with RE Xho1 and Nde1 (see lanes 5 and 6). Expected size ~2600 bp. Both samples had postive inserts, sent sample 1 (lane 5) to sequencing<br> <br />
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Date:&nbsp;June 16, 2012<br> <br />
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[[Image:1137 0716 Screening.JPG]]<br> <br />
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Sequencing came back bad, grow more bacteria from plate, redid MiniPrep, digested plasmids with RE Xho1 and Nde1. Expected size ~2600 bp. All? samples had postive inserts, sent sample 2 (lane 2) to sequencing<br> <br />
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Date:&nbsp;July 17, 2012 <br />
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[[Image:0717 Digestion.JPG]] <br />
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Digested sample 2 again with Xho1 and Nde1 (see lane 3), excepted size 2600 bp. Too many bands? <br />
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Date:&nbsp;July 23, 2012<br><br />
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Prepared MiniPrep, digested plasmids with RE Xho1 and Nde1. Expected size ~2600 bp. Samples 1, 3, 4, 5 and 6 had postive inserts, sent sample 1 (lane 1) to sequencing<br><br />
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[[Image:1137_0723_Screening.JPG]]<br />
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== '''Result intepretation/Summary''' ==<br />
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== '''Conclusion''' ==<br />
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[[Category:Cloning]]</div>
Paul.xu