Team:Yale/Our Project/Notebook/Week 9

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Revision as of 03:12, 28 October 2010

iGEM Yale

our project

lab notebook: week 9

Monday 8/2--Analysis of second ligation transformants

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 <li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li>
 <li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li>
-<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></b></li>
+<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li>
 <li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li>
 <li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li>
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+<h4> Work with transformants from attempt #1 to complete second ligation </h4>
+<ul>
+<li>Chose seven colonies from the 2:1 insert:vector ligation plate (colonies #1-#7) and seven from the 5:1 ligation plate (colonies #8-#14) to analyze.  Used each to inoculate a liquid culture, streak an index plate, and run a colony PCR reaction (after appropriate lysing in water).</li>
+<li>Each PCR reaction mixture contained 2 uL of VF2 primer, 2 of VR primer, 9.8 uL of water, 4 uL of Phusion buffer, 0.6 uL of DMSO, 0.4 uL of dNTPs, and 0.2 uL of polymerase.  They were run on the 'VR' thermocycler protocol</li>
+<li> The PCR products were then run on a 1.0% agarose gel at 90 V vs. a 1 kb ladder.</li>
+Loaded ladder at the far left and samples 1 through 14 from left to right in order.  Only sample 6 is even close the size range of the desired ligation product (8.3 kb) and even it is still at least a kb too small to be the right construct. <br/>
 <i> This day's labwork is also recorded on pages 85 and 86 of the hard copy lab notebook. </i>
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