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456 pLex-CMV-CuO-Dest - Revision history
2026-10-18T08:46:59Z
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Luyingying at 20:33, 28 November 2011
2011-11-28T20:33:35Z
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<p><b>New page</b></p><div>Cloning of pLex-CMV-CuO-Dest (456) <br />
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Purpose: to clone pLex-CMV-CuO-Dest by PCR <br />
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Date: 2011-11-3 to 2011-11- <br />
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Notebook: Xiaojing Wang <br />
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Page: 004113---004114 <br />
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Clone ID: 456 <br />
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Experimenters: Xiaojing Wang <br />
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Results <br> <br />
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[[Image:456 PCR 11.3.2011.JPG|141x285px|456 PCR 11.3.2011.JPG]]<br> <br />
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The bands show PCR product of 456, <br />
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Primer: <br />
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GW-SpeI-F agtac ACTAGT ATCAACAAGTTTGTACAAAAAAG <br />
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GW-Mlu1-R (CTTTCTTGTACAAAGTGGTTGAT CAC CAt CAC CAt CAC CAt tga ACGCGT atcta ) tagatACGCGTtcaaTGGTGaTGGTGaTGGTGATCAACCACTTTGTACAAGAAAG <br />
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Template is 392 pLVB-V5-Dest <br />
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Digest result:&nbsp; <br> <br />
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[[Image:456 PCR digest 11.3.2011.JPG|155x381px|456 PCR digest 11.3.2011.JPG]]<br> <br />
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digest PCR product and vector with Spe1 + Mlu1,run gel <br />
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gel purify, ligate, transform ccdb survival <br />
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<br>Colony Screening <br> <br />
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[[Image:456 clone digest 11.11.2011.JPG]] <br />
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Select 5 clones to mini-prep, digestd by Spl I and Mlu I.&nbsp; 456-1,-3.-4 may be correct. <br> <br />
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pDonr221_EGFP(393) +&nbsp;pLex-CMV-CuO-Dest(456-1/456-3/456-4) &nbsp; make gateway by LR Clonase II.<br> <br />
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Transform 393+456-1/456-3/456-4 by STBL3, Amp.<br> <br />
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Select 4 clones each plate to mini-prep. The total number of plasmids is 12 (No.1-12). <br />
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[[Image:393+456 gateway by xiaojing 11.18.2011.jpg]] <br />
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No.1-12 plasmids transfect 293T cell line.<br> <br />
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&nbsp;48 hours after transfection, take pictures. <br />
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[[Image:393+456 Digest 11.18.2011.JPG]] <br />
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No.1-12 plasmids are digested by BamHI. Bands 13 and 14 are control. <br />
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Band 13 is 456-3 digested by BamHI, and band 14 is 456-3. <br />
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No.1-5,7-9,11,12 may be correct. <br />
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Date: 2011-11-7 to 2011-11-10 <br />
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Notebook: Yingying Lu<br> <br />
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Page: <br> <br />
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Clone ID: 456 <br />
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Experimenters: Yingying Lu<br> <br />
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Results <br> <br />
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[[Image:456 pcr11-7-2011.JPG]]<br> <br />
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GW-SpeI-F agtac ACTAGT ATCAACAAGTTTGTACAAAAAAG <br />
<br />
GW-Mlu1-R (CTTTCTTGTACAAAGTGGTTGAT CAC CAt CAC CAt CAC CAt tga ACGCGT atcta ) tagatACGCGTtcaaTGGTGaTGGTGaTGGTGATCAACCACTTTGTACAAGAAAG <br />
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Template is 392 pLVB-V5-Dest <br> <br />
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[[Image:456+493 clone11-8-2011.JPG]]<br> <br />
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digest PCR product and vector448pLex-CMV-CuO with Spe1 + Mlu1,run gel <br> <br />
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band 1 show insert&nbsp; ,band 2 show vector<br> <br />
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[[Image:418+419+456 digest detect11-10-2011.JPG]]<br> <br />
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5 clonies digest detect:Spe1 + Mlu1, 456-3,456-5 may be right<br> <br />
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[[Image:393+456 gateway by yingying 11.18.2011.jpg]] <br />
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393EGFP to 456-5&nbsp; 5 clonies&nbsp; transfect into cells <br> <br />
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[[Image:393+456 digest detect11-16-2011.JPG]]<br> <br />
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393-pDonr221_EGFP to 456-5 pLex-CMV-CuO-Dest <br> <br />
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then digest detect with BamH1<br> <br />
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Band 1---5 show 5 clones, band 6 show 456-5 pLex-CMV-CuO-Dest <br> <br />
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[[Category:Cloning]]</div>
Luyingying