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1206 pET28b-recT - Revision history
2026-10-18T03:05:59Z
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Paul.xu at 16:23, 23 July 2012
2012-07-23T16:23:02Z
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<p><b>New page</b></p><div>== '''Cloning of 1206 pET28b-recT''' ==<br />
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'''Purpose:''' Cloning of 1206 pET28b-recT <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: 1206''' <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 3 (expected size = 834 bp), purified and digested wth vector 12 (pET28b)&nbsp;with restriction enzymes Xho1 and Nhe1<br> <br />
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[[Image:0626 Digestion.JPG]]<br> <br />
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Cut out insert from lane 3 and vector from lane 4, purified and ligated overnight. Transform with Top10 cells and plate on&nbsp;KAN&nbsp;plates tomorrow <br />
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Date:&nbsp;July 18, 2012<br> <br />
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Cut out band in lane 1 and 2 (expected size = 834 bp), purified and digested wth vector 12 (pET28b) with restriction enzymes Xho1 and Nhe1<br> <br />
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[[Image:1209 0718 Amp.JPG]] <br />
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Cut out insert from lane 1 and vector from lane 2, purified and ligated overnight. Transform with Top10 cells and plate on KAN plates tomorrow <br> <br />
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[[Image:1206 0718 Digestion.JPG]] <br />
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=== <u></u>Colony Screening ===<br />
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[[Image:1206 Screening.JPG]]<br> <br />
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Screened colonies, all positve, expected ize ~800 bp <br />
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Grew lanes/sample 1-6 in media in shaker overnight, MiniPrep tomorrow. <br />
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Date:&nbsp;June 29, 2012 <br />
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[[Image:0629 Screening.JPG]] <br />
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Digested MiniPrep with Nhe1 + Xho1, sent sample 3 (lane 9)&nbsp;to sequencing <br />
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'''looks like the digestion is not&nbsp; complete (two big bands), need to digest a little bit longer or add more enzyme''' <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 Nhe1 (see lanes 3 and 4). Expected size ~800 bp. Both samples had postive inserts, sent sample 2 (lane 4) to sequencing<br> <br />
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Date:&nbsp;July 16, 2012<br> <br />
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[[Image:1206 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 Nhe1. Expected size ~800 bp. All samples had postive inserts?<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 4 with Xho1 and Nhe1 (see lane 4), expected size ~800 bp. Too many bands <br />
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Date: July 20, 2012 <br />
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Screened colonies for positive inserts <br />
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[[Image:1206 0720 Screening.JPG]] <br />
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Grew samples 1, 2, 3, 5, 6 and 7 in media and put in shaker overnight <br />
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Date:&nbsp;July 23, 2012<br><br />
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[[Image:1206_0623_Screening.JPG]]<br><br />
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Prepared MiniPrep, digested plasmids with RE Xho1 and Nhe1. Expected size ~800 bp. All samples had postive inserts, sumbitted sample 6 (lane 6)&nbsp;for sequencing<br><br />
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== '''Result intepretation/Summary''' ==<br />
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== '''Conclusion''' ==<br />
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[[Category:Cloning]]</div>
Paul.xu