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1148 pTXB1-CH - Revision history
2026-10-18T02:39:40Z
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Xiaojing.Wang at 16:46, 20 July 2012
2012-07-20T16:46:29Z
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<p><b>New page</b></p><div>== '''Cloning of 1148 pTXB1-CH''' ==<br />
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'''Purpose:''' Cloning of 1148 pTXB1-CH <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: 1148''' <br />
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'''Experimenters: '''Jennifer Truong <br />
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== '''Results''' ==<br />
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=== Cloning ===<br />
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[[Image:0626 Amp.JPG]]<br> <br />
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Amplified DNA&nbsp;with PHUSION&nbsp;enzyme (0.5 uL , 25 cycles, 55 degees annealing temperature)<br> <br />
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Cut out band in lane 2 (expected size = 784 bp), purified and digested wth vector 1126 (pTXB1)&nbsp;with restriction enzymes Xho1 and Pst1<br> <br />
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[[Image:0626 Digestion.JPG]] <br />
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I&nbsp;don't know what happened to the digested insert (lane 1), will redo tomorrow. Cut out digested vector (lane 2) to save <br />
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Date:&nbsp;June 27, 2012<br> <br />
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Reamplified DNA with PHUSION enzyme (0.5 uL , 25 cycles, 55 degees annealing temperature) <br />
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Cut out band in lane 3 (expected size = 784 bp), purified and digested wth vector 1126 (pTXB1) with restriction enzymes Xho1 and Pst1<br> <br />
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[[Image:0627 amp.JPG]] <br />
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Digested 1148 DNA&nbsp;and pTXB1 vector with restriction enzymes Xho1 and Pst1 <br />
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[[Image:0627 Digestion.JPG]] <br />
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See lanes 2 and 3. Cut out insert from lane 2, and cut out top vector band in lane 3. Purified and ligated together. Transformed with Top10 cells, plated on Amp resistant plates, grow overnight. <br />
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Date:&nbsp;July 18, 2012<br> <br />
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Started all over becuase this experiment was started before the power went out. Samples may have problems<br> <br />
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[[Image:1148 0718 Amp.JPG]]<br> <br />
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Cut out band in lanes 1 and 2 (expected size = 784 bp), purified and digested wth vector 1126 (pTXB1) with restriction enzymes Xho1 and Pst1<br><br />
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[[Image:1148_Digestion.JPG]]<br><br />
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See lanes 1 and 2. Cut out insert from lane 1, and cut out top vector band in lane 2. Purified and ligated together. Transformed with Top10 cells, plated on Amp resistant plates, grow overnight. <br><br />
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=== Colony Screening ===<br />
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[[Image:1148 Screening.JPG]]<br> <br />
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Screened colonies. <br />
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Samples 1, 2, 3, 4, 6 and 7 were positive (same respected lanes), expected size ~800 bp <br />
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Grew in media in shaker overnight. <br />
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[[Image:0629 Screening.JPG]] <br />
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Screening was bad, redo <br> <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 Pst1 (see lanes 1 and 2). Expected size ~800 bp. Both samples had postive inserts, sent sample 1 to sequencing<br> <br />
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Date: July 16, 2012<br> <br />
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[[Image:1149 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 Pst1. Expected size ~800 bp. All samples had postive inserts?<br> <br />
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July 17, 2012<br> <br />
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Digested sample 4, see lane 5 again with Xho1 and Pst, expected size ~800 bp. No insert <br> <br />
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[[Image:0717 Digestion.JPG]]<br> <br />
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Date: July 19, 2012<br> <br />
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[[Image:1148 0719 Screening.JPG]] <br />
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Samples 1, 2, 3, 4, 5 and 6 were positive (same respected lanes), expected size ~800 bp <br />
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Grew in media in shaker overnight. <br> <br />
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Date&nbsp;:&nbsp;July 20, 2012<br> <br />
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Digested MiniPrep plasmids with RE Xho1 and Pst1. Expected size ~800 bp. All samples had postive inserts?&nbsp;Sent sample 1 to sequencing<br> <br />
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[[Image:1148_0720_Digestion.JPG]]<br />
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== '''Result intepretation/Summary''' ==<br />
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== '''Conclusion''' ==<br />
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[[Category:Cloning]]</div>
Xiaojing.Wang