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1154 pCAMBIA-2301-Ara-s1 - Revision history
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Jennifer.truong: /* Colony Screening */
2012-06-05T01:25:21Z
<p><span class="autocomment">Colony Screening</span></p>
<p><b>New page</b></p><div>== '''Cloning of Arabidopsis S1 Nuclease into pCAMBIA-2301''' ==<br />
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'''Purpose:''' Cloning of&nbsp;Arabidopsis S1 Nuclease into pCAMBIA-2301 <br />
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'''Date:&nbsp;''' 05/24/2012<br> <br />
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'''Notebook:&nbsp;''' <br />
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'''Page: 008803-003308''' <br />
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'''Clone ID: 1154''' <br />
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'''Experimenters:'''&nbsp; Jennifer Truong<br> <br />
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== '''Cloning''' ==<br />
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Date: 05/24/2012<br> <br />
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PCR Template: <br />
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[[Image:1154 PCR 2012 4 25.jpg]] <br />
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Isolated band for cloning next week <br />
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Missing template gel<br />
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== '''Colony Screening'''<br> ==<br />
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[[Image:1154 PCR Screening 05302012.JPG]]<br> <br />
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PCR Screening showed 100% efficency, grow samples 1-6 (lanes)&nbsp;in medium<br> <br />
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Prepared Miniprep for samples 1-6, digested incorrect enzymes BglII and Nhe1<br />
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[[Image:1154 Digest FAIL.JPG]] <br />
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Submitted 1154-4 for sequencing, yielded by sequencing results, redid digest with&nbsp;Xho1 and Nco1<br />
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[[Image:1154 Digest 1.JPG]] <br />
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Digest successfully shows insert in all samples, submitted 1154-1 for sequencing<br />
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6/4/2012: 1154 turned on mutated, submitted 1154-5 for sequencing.<br />
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== '''Result intepretation/Summary''' ==<br />
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Summary:<br> <br />
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Oligomix 1:&nbsp;1 uL of 10uM 1-14 <br />
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Oligomix 2:&nbsp;1 uL&nbsp;of 10mM 13-26 <br />
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Reaction mixture:&nbsp;0.5uL of oligomix 0.25uL Kapa HiFi polymerase <br />
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5uL 10x PCR&nbsp;Buffer <br />
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1uL of 10mM dNTP <br />
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18.25uL&nbsp;of water <br />
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PCR&nbsp;at Kapa setting 10 cycles with 30 seconds extension <br />
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Combined both reaction mixtures after PCR , add 1uL of 10mM Ara-S1-1 and 1uL of 10mM Ara-S1-26. PCR&nbsp;setting 25 cycles with 30 seconds extension. <br />
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Added 10.0 uL of 6x dye to PCR&nbsp;sample, load ~15 uL&nbsp;of sample into each well (lane one and two), loaded 5 uL of 1 kB buffer, ran gel in 0.5x buffer 15 minutes. <br />
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== '''Conclusion''' ==<br />
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No results because the PCR lid was not on tight enough, solution evaporated, compromising the Mg composition of the solution resulting in all of the DNA&nbsp;to not be digested and remained in the well. <br> <br />
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5/30/2012:&nbsp;After redoing the PCR, was able to succesfully transform the cells that were grown on the media.<br> <br />
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[[Category:Cloning]]</div>
Jennifer.truong