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193-pCMVK-StrepII - Revision history
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Paul.xu at 00:01, 5 September 2011
2011-09-05T00:01:53Z
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<p><b>New page</b></p><div>= '''pCMVK-StrepII (193)'''<br> =<br />
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== '''Make&nbsp; pCMVK-StrepII''' ==<br />
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'''Date: 2011-09-02''' <br />
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'''Notebook:''' <br> <br />
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'''Page:''' <br> <br />
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'''Clone ID:''' 193 <br />
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'''Experimenters:''' <span style="font-weight: bold;">Paul</span><br> <br />
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=== Annealing ===<br />
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Date: <br> Anneal 8685-T and 8685-B at 95<sub><sup>0</sup></sub>C for 2 minutes. <br> Digest the vector pCMVK-GFP(#194) with BgIII+ Sal1<br> <br />
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Run gel and cut out vector,&nbsp; purify using spin column <br />
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Ligate with annealed oligos for 2 hours<br> <br />
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Primer-F 8685-T tc gac CACGTG GAATTC TCTAGA TGG AGC CAC CCG CAG TTC GAA AAG TAA CTC GAG GGATCC A<br>Primer-R 8685-B GATCTGGATCCCTCGAGTTACTTTTCGAACTGCGGGTGGCTCCATCTAGAGAATTCcacgtgg<br> <br />
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and transform Top10 <br />
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[[Image:PCMVK-StrepII-08-30-2011-1.JPG]]<br> <br />
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Lane 1 shows the digestion of vector (pCMVK-GFP) with BgIII+ Sal1;<br> <br />
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Lane 2: the plasmid F2 from # 7896 Kinase, digested with Sal1 + HindIII <br />
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Lane 3: the plasmid F02 from # 7898 Kinase, digested with Sal1 + HindIII <br />
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Lane 4:digestion of #348 <br />
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Lane 5: PlentiGP-app, run DNA Argrose gel after miniprep <br />
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== Colony Screening by Restriction Digestion<br> ==<br />
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1, 2,: Digest with BglII + KpnI <br />
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3, 4, 5: 194 digest with BglII + Kpn1 <br />
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6~10: 193 digestion with BglII + Kpn1 <br />
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11, 12, 13: 194 digest with Kpn1+ Xho1 <br />
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14~18: 193 digest with Kpn1 + Xho1 <br />
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[[Image:192 193 194 digestion 94 2011.jpg]] <br />
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[[Image:192 193 194 digestion 9 4 2011 2.jpg]] <br />
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193: digest with Kpn1+ EcoR1 <br />
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[[Image:193 Kpn1 EcoR1 9 4 2011.jpg]] <br />
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[[Image:193 Kpn1 EcoR1 9 4 2011 2.jpg]] <br />
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[[Image:193 Kpn1 Xho1 KpnI BamH1.jpg]]<br> <br />
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digest with Kpn1 + Sal1<br />
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[[Image:193_Kpn1_Sal1_TaqMuts.jpg]]<br />
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Looks like clone #4 is good. <br />
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--[[User:Paul.xu|Paul.xu]] 15:44, 4 September 2011 (EST) <br />
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[[Category:Cloning]]</div>
Paul.xu