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1283 pDB.His.MBP.Ent - Revision history
2026-10-18T05:41:43Z
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Jennifer.truong at 20:16, 20 August 2012
2012-08-20T20:16:18Z
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<p><b>New page</b></p><div>== '''Cloning of 1283 pDB.His.MBP.Ent''' ==<br />
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'''Purpose:''' Cloning of 1283 pDB.His.MBP.Ent <br />
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'''Date: '''July 31, 2012<br> <br />
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'''Notebook:&nbsp;''' <br />
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'''Page:''' <br />
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'''Clone ID: '''1283 <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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Date:&nbsp;July 31, 2012<br> <br />
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[[Image:0731 Amp.JPG]]<br> <br />
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PCR MBP from pMAL-p5E using<br>1 ul of 10 uM MBP-Xba1-F1 cgatcgTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATAatgggcagcagccatc<br>1 ul of 1 uM MBP-F2 GATATAatgggcagcagccatcatcatcatcatcacATGAAAATCGAAGAAGGTAAAC <br>1 ul of 10 uM MBP-Nde1-R gtgctatCATATGGCCCATGGACGGTACCTTGTCATCGTCATCCCCGAGGTTGTTGTTATTGTTATTGTTG <br />
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Digest PCR product with Xba1 + Nde1, ligate to Xba1 + Nde1 digested vector pDB.His.BMP, V14<br> <br />
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See lane 2, expected size ~1400 bp<br> <br />
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Cut out band and digested <br> <br />
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[[Image:1283 Digestion.JPG]]<br> <br />
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Digest PCR product with Xba1 + Nde1 (lane 1), ligate to Xba1 + Nde1 digested vector pDB.His.BMP, V14 (lane 2)&nbsp;<br> <br />
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Cut out bands, purify and ligate together overnight <br />
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Date:&nbsp;August 7, 2012<br> <br />
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[[Image:1283 0807 Amp.JPG]]<br> <br />
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Sequencing came back contaminated, started all over<br> <br />
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See lanes 1 and 2<br> <br />
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PCR MBP from pMAL-p5E using<br>1 ul of 10 uM MBP-Xba1-F1 cgatcgTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATAatgggcagcagccatc<br>1 ul of 1 uM MBP-F2 GATATAatgggcagcagccatcatcatcatcatcacATGAAAATCGAAGAAGGTAAAC<br>1 ul of 10 uM MBP-Nde1-R gtgctatCATATGGCCCATGGACGGTACCTTGTCATCGTCATCCCCGAGGTTGTTGTTATTGTTATTGTTG <br />
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Digest PCR product with Xba1 + Nde1, ligate to Xba1 + Nde1 digested vector pDB.His.BMP, V14 <br />
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See lane 2, expected size ~1400 bp <br />
<br />
Cut out band and digested <br> <br />
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[[Image:1283 0810 Digestion.JPG]]<br> <br />
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Digest PCR product with Xba1 + Nde1 (lane 1), ligate to Xba1 + Nde1 digested vector pDB.His.BMP, V14 (lane 2) <br />
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Cut out bands, purify and ligate together overnight <br> <br />
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Date:&nbsp;August 9, 2012 <br />
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Digestion was messed up from earlier because of the gel, started all over <br />
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[[Image:1283 0809 Amp.JPG]] <br />
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Cut out lanes 1 and 2 for digestion, save 3 and 4 in box just incase something happens <br />
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Date:&nbsp;August 17, 2012<br> <br />
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[[Image:1283 0817 Digestion.JPG]]<br> <br />
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Digest PCR product with Xba1 + Nde1 (lane 7), ligate to Xba1 + Nde1 digested vector pDB.His.BMP, V14 (lane 8) <br />
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Cut out bands, purify and ligate together overnight <br> <br />
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=== <u></u>Colony Screening ===<br />
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Date:&nbsp;August 3, 2012<br> <br />
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[[Image:1283 Screening.JPG]]<br> <br />
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Digested MiniPrep plasmids with RE Xba1 +&nbsp;Nde1 in lanes 1 - 6 (samples 1-6 respectively)<br> <br />
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Digested sample 1 with Xba1 +&nbsp;Nco1 in lane 8, and digested sample 1 with Xba1 +&nbsp;Kpn1 in lane 9 <br />
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All correct sizes, send sample 1 to sequencing<br />
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Clones <br><br> <br />
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'''These are contaminated clones''' <br />
Date:&nbsp; 2012<br />
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[[Image:1283_0820_Screening.JPG]]<br />
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Clones 2, 3, 4, 5 and 6 may be correct<br />
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== '''Result intepretation/Summary''' ==<br />
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== '''Conclusion''' ==<br />
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[[Category:Cloning]]</div>
Jennifer.truong