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1380 pCMV5-Fc-(3c)-GNE-WPRE-oriP - Revision history
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Xiuli.huang at 14:58, 25 September 2012
2012-09-25T14:58:08Z
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<p><b>New page</b></p><div>== '''1380 Cloning of pCMV5-Fc-(3c)-GNE-WPRE-oriP''' ==<br />
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'''Purpose:''' subcloning of gene GNE from 1269 pCMV5-Fc(3c)-GNE into 1369 pCMV5-Fc-(3c)-TagRFP-WPRE-oriP with NheI and NotI<br />
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'''Date:''' 2012-09-20 <br />
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'''Notebook:'''<br />
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'''Clone ID: 1380''' <br />
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'''Experimenters: Xiuli''' <br />
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== '''Results''' ==<br />
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Cloning <br />
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[[Image:9202012-1377-1381.jpg]]&lt;br&gt; <br />
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09-20-2012 <br />
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A.Restriction enzyme digestion: plasmid 1284,1276 1264, 1269, 1263 and 1396 cut with NheI and NotI.&lt;br&gt; <br />
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B. Gel purification and elute with 15 ul H2O <br />
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c Ligation: 2xLigation buffer (12ul) +12 ul elution from above +0,5 ul T7 ligase. Incubate at RT for 1 hour&lt;br&gt; <br />
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D. Transformation.<br> 9/22/2012 <br />
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make miniprep, digest with Nhe1 + Not1 <br />
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[[Image:1377 78 79 80 81 2012 9 22.jpg]] <br />
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[[Image:1377 78 79 80 81 2012 9 22 2.jpg]] <br />
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9/24/2012 <br />
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Select 1377-2, 1378-1, 1379-2, 1380-2, 1381-1 to make maxiprep using Qiagen maxiprep kit. 1381, 1392&nbsp; using invitrogen maxiprep&nbsp; kit <br />
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[[Image:1377-1381 maxi cut .jpg]][[Image:1377-1381 maxi-cut.jpg]] <br />
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1381 and 1392 using Invitrogen maxi prep kit have too much RNA. After treating&nbsp; sample 1381 and 1392 with 0.5 ul RNAse (Qiagen) at 37oC for 2-3 hours, precipitate DNA&nbsp;with isoprapanol. <br />
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[[Category:Cloning]]</div>
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