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1118 p8H-Klenow - Revision history
2026-10-18T06:28:24Z
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Paul.xu at 17:30, 17 December 2012
2012-12-17T17:30:24Z
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<p><b>New page</b></p><div>== '''Cloning of 1118 p8H-Klenow''' ==<br />
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'''Purpose:''' Cloning of 1118 p8H-Klenow <br />
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'''Date: '''July 23, 2012<br> <br />
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'''Notebook:&nbsp;''' <br />
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'''Page:''' <br />
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'''Clone ID: '''1118 <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 24, 2012<br> <br />
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[[Image:0724 Amp.JPG]]<br> <br />
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See lane 1, expected size 1817 bp <br> <br />
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PCR using MG1665 genomic DNA as template<br>DNA-Pol1-Sal1-R<br>(cgaaaactgggatcaggcgcac GTC GAC tca cgat)<br>atcgtgaGTCGACGCGGCCGCCgtgcgcctgatcccagttttcg <br />
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Klenow-Nde1-F<br>cagtcg catatg GTGATTTCTTATGACAACTAC<br> <br />
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Cut out band, purify, digest with RE&nbsp;Ndel and Sal1 with vector 1135 <br />
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[[Image:0726 Digestion.JPG]] <br />
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See lanes 4 (insert)&nbsp;and 5 (vector) <br />
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Lost insert, need to reamplify, save the digested vector <br />
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[[Image:0724 Amp 2.JPG]]<br> <br />
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See lane 1, expected size 1817 bp <br />
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PCR using MG1665 genomic DNA as template<br>DNA-Pol1-Sal1-R<br>(cgaaaactgggatcaggcgcac GTC GAC tca cgat)<br>atcgtgaGTCGACGCGGCCGCCgtgcgcctgatcccagttttcg <br />
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Klenow-Nde1-F<br>cagtcg catatg GTGATTTCTTATGACAACTAC <br />
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Cut out band, purify, digest with RE Ndel and Sal1<br> <br />
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[[Image:0724 Digestion.JPG]] <br />
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Digested with Nde1 and Sal1 (see lane 1), cut out band, purify and ligate together with vector saved form earlier. Ligate over night, transform tomorrow morning <br />
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Date:&nbsp;August 2, 2012<br> <br />
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Screening came back bad, started all over<br> <br />
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[[Image:0802 Amp.JPG]]<br> <br />
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See lane 2, expected size 1817 bp <br />
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PCR using MG1665 genomic DNA as template<br>DNA-Pol1-Sal1-R<br>(cgaaaactgggatcaggcgcac GTC GAC tca cgat)<br>atcgtgaGTCGACGCGGCCGCCgtgcgcctgatcccagttttcg <br />
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Klenow-Nde1-F<br>cagtcg catatg GTGATTTCTTATGACAACTAC <br />
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Cut out band, purify, digest with RE Ndel and Sal1 with vector 1135 <br> <br />
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[[Image:1118 Digestion.JPG]]<br> <br />
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See lanes 3 (insert) and 4 (vector), cut out, ligate together, transform<br> <br />
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=== <u></u>Colony Screening<br> ===<br />
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Date:&nbsp;July 27, 2012 <br />
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[[Image:0727 Digestion.JPG]] <br />
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Digested MiniPrep with RE&nbsp;Nde1 and Sal1, expected size 1817 bp. <br />
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Sample 4 may be correct <br />
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Date:&nbsp;August 6, 2012<br> <br />
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[[Image:1118 0806 Screening.JPG]]<br> <br />
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Digested MiniPrep with RE Nde1 and Sal1, expected size 1817 bp. <br> <br />
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No inserts present<br> <br />
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<br> <br />
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'''Cloning of 1118 pET28b.Amp-Klenow''' <br />
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Purpose: Cloning of 1118 pET28b.Amp-Klenow <br />
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Date: Dec, 2012 <br />
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Notebook: <br />
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Page: <br />
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Clone ID: 1118 <br />
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Experimenters: Shanyi<br> <br />
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<br>'''Results'''<br> <br />
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'''[[Image:1118PCR.JPG]]'''<br> <br />
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PCR using DH5alpha genomic DNA as template<br> <br />
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'''[[Image:1118PCR 1422+1197 1429+1207 1279DIGEST.JPG]]<br>'''digest 1422and 1118pcr products<br> <br />
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[[Image:1118PCR 1422 1240PCR V12 1505DIGEST.JPG]] <br />
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screen<br />
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[[Image:1118_1240digest.JPG]]<br />
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== '''Result intepretation/Summary''' ==<br />
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== '''Conclusion''' ==<br />
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<br> <br />
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[[Category:Cloning]]</div>
Paul.xu