Team:Yale/Our Project/Notebook/Week 8

From 2010.igem.org

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<!------------- atgc ------------->
<!------------- atgc ------------->
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>sequence of desired phsABC/B0015 construct (in
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>sequence of desired phsABC/B0015 construct  
<!------------- atgc ------------->
<!------------- atgc ------------->
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<h4>Beginning of second ligation efforts </h4>
<h4>Beginning of second ligation efforts </h4>
<ul>  
<ul>  
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<li>With the phsABC insert finally in B0015, the next step is to insert the Q04121 promoter in front of phsABC. Begin with sequential double digests of Q04121 and the phsABC/B0015 construct</li>
+
<li>With the phsABC insert finally in B0015, the next step is to insert the Q04121 promoter in front of phsABC. Begin with double digests of Q04121 and the phsABC/B0015 construct double digest</li>
 +
<li>Q04121 double digest: 6 uL of Q04121, 5 uL of EcoRI buffer, 1.8 uL of EcoRI, 1.8 uL of SpeI, 0.5 uL of 100x BSA, and 34.9 uL of water.  </li>
 +
<li>phsABC/B0015 construct sequential digest: 5 uL of DNA, 5 uL of NEB buffer 4, 0.5 uL of BSA, 1.8 uL of XbaI, and 37.7 uL of water.</li>
 +
<li> Run both of the digests for six hours at 37˚C before heat-killing with 20 minutes at 80˚C, then add 0.5 uL 5 M NaCl and 1.8 uL of EcoRI to the vector and let it have a five hour digestion period at 37˚C followed by another heat-kill at 80˚C. </li>
</ul>
</ul>
<i> This day's work is also recorded on page 83 & 84 of the lab notebook hard copy. </i>
<i> This day's work is also recorded on page 83 & 84 of the lab notebook hard copy. </i>
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<li>
<li>
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Thursday 7/29--
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Thursday 7/29--First attempt at Q04121 ligation
</li>
</li>

Revision as of 01:49, 28 October 2010

iGEM Yale

lab notebook: week 8 (7/26-8/1)

  • Monday 7/26--Efforts to confirm that ligation of phsABC and B0015 actually occurred.
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  • Tuesday 7/27--Diagnostic double digest of likely ligations
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  • Wednesday 7/28--Sequencing confirms ligation success!
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  • Thursday 7/29--First attempt at Q04121 ligation
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  • Friday 7/30--
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  • Saturday short content
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  • Sunday short content
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