Difference between revisions of "Team:UCLA/Notebook/Spider Silk Genetics/7 May 2015"

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<html><head><title>5/7/2015</title><meta content="text/html; charset=UTF-8" http-equiv="content-type"><style type="text/css">ol{margin:0;padding:0}.c0{widows:2;orphans:2;direction:ltr}.c3{max-width:468pt;background-color:#ffffff;padding:72pt 72pt 72pt 72pt}.c2{font-weight:bold}.c1{height:11pt}.title{widows:2;padding-top:0pt;line-height:1.15;orphans:2;text-align:left;color:#000000;font-size:21pt;font-family:"Trebuchet MS";padding-bottom:0pt;page-break-after:avoid}.subtitle{widows:2;padding-top:0pt;line-height:1.15;orphans:2;text-align:left;color:#666666;font-style:italic;font-size:13pt;font-family:"Trebuchet MS";padding-bottom:10pt;page-break-after:avoid}li{color:#000000;font-size:11pt;font-family:"Arial"}p{color:#000000;font-size:11pt;margin:0;font-family:"Arial"}h1{widows:2;padding-top:10pt;line-height:1.15;orphans:2;text-align:left;color:#000000;font-size:16pt;font-family:"Trebuchet 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class="c3"><p class="c0"><span>Performed a Ligation/Digestion test based on golden gate cloning, reactions as follows:</span></p><p class="c0 c1"><span></span></p><p class="c0 c1"><span></span></p><p class="c0"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Tube 1&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Tube 2 (control)</span></p><p class="c0"><span>2AB (380 ng/uL)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2.63 uL&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;0 uL</span></p><p class="c0"><span>2BC (460 ng/uL)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2.17 uL&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4.34 uL</span></p><p class="c0"><span>10x T4 Ligase buffer&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2 uL&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2 uL</span></p><p class="c0"><span>BSA (1mg/mL)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2 uL&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2 uL</span></p><p class="c0"><span>BsaI (15 U)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1.5 uL&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1.5 uL</span></p><p class="c0"><span>T4 Ligase (5 U)*&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1.25 uL&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1.25 uL</span></p><p class="c0"><span>ddH20&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;8.45 uL&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;8.91 uL</span></p><p class="c0"><span class="c2">Total&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;20 uL&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;20 uL</span></p><p class="c0 c1"><span></span></p><p class="c0"><span>37 &#8451;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;20 s</span></p><p class="c0"><span>37 &#8451;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2 min</span></p><p class="c0"><span>*</span></p><p class="c0"><span>16 &#8451; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3 min</span></p><p class="c0"><span>50 &#8451;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5 min</span></p><p class="c0"><span>*Repeat from * 30 x</span></p><p class="c0 c1"><span></span></p><p class="c0"><span>80 &#8451;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5 min</span></p><p class="c0"><span>12 &#8451;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;hold</span></p><p class="c0"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></p><p class="c0"><span>*T4 Ligase was diluted 1:100 in 1x T4 Ligase buffer prior to adding 1.25 uL</span></p><p class="c0"><span>1 ug of each plasmid was used.</span></p><p class="c0 c1"><span></span></p><p class="c0"><span>Visualized Ligation/Digestion Test on 2% TAE gel. Results indicate that digestion occurred ~50% efficiency. The undigested and digested backbones appeared at approx. equal intensity. No bands smaller than 1000 bp appeared, cannot say for sure whether ligation took place. </span></p><p class="c0 c1"><span></span></p><p class="c0"><span>Gel purified BsaI digested plasmids from yesterday. Yields: ~7 ng/uL for AB, BC, CA.</span></p></body></html>
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<h2 class="titleHead">5/7/2015</h2>
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  <h3 class="sectionHead"><span class="titlemark">1  </span> <a
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id="x1-10001"></a>BsaI Digestion of 2AB, 2BC, 2CA</h3>
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  Gel purification of yesterday&#8217;s BsaI digested fragments (Qiagen).
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  Obtained approx. 7 ng/uL for each monomer. A 260/280 for all three were
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around 1.8. Will use these as is for ligation test tomorrow.
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Latest revision as of 05:17, 16 May 2015

iGEM UCLA




5/7/2015

5/7/2015


1 BsaI Digestion of 2AB, 2BC, 2CA

∙ Gel purification of yesterday’s BsaI digested fragments (Qiagen).

∙ Obtained approx. 7 ng/uL for each monomer. A 260/280 for all three were around 1.8. Will use these as is for ligation test tomorrow.