Team:Yale/Our Project/Notebook/Week 7

From 2010.igem.org

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<b> Dual-purpose gel </b> <br/>
<b> Dual-purpose gel </b> <br/>
<li> Ran a gel to accomplish two things: 1.Determine whether the phsABC fragment was still present after gel extraction 2. Determine based on dimer formation of singly digested phsABC fragments whether the digestion/ligation is working well.  In a 1.0% agarose gel run at 90 V had the following lane assignments, left to right: Lane 1--1 kb ladder, Lane 2--circular 8 kb pSB74 plasmid, Lane 3--1 kb ladder, Lane 4--phsABC from gel extraction, Lane 5--self ligation of SpeI-digested phsABC (iGEM ligase), Lane 6--self ligation of SpeI-digested phsABC (Grindley lab ligase), Lane 7--self ligation of EcoRI-digested phsABC (iGEM ligase), Lane 8--self ligation of EcoRI-digested phsABC (Grindley lab ligase)</li>
<li> Ran a gel to accomplish two things: 1.Determine whether the phsABC fragment was still present after gel extraction 2. Determine based on dimer formation of singly digested phsABC fragments whether the digestion/ligation is working well.  In a 1.0% agarose gel run at 90 V had the following lane assignments, left to right: Lane 1--1 kb ladder, Lane 2--circular 8 kb pSB74 plasmid, Lane 3--1 kb ladder, Lane 4--phsABC from gel extraction, Lane 5--self ligation of SpeI-digested phsABC (iGEM ligase), Lane 6--self ligation of SpeI-digested phsABC (Grindley lab ligase), Lane 7--self ligation of EcoRI-digested phsABC (iGEM ligase), Lane 8--self ligation of EcoRI-digested phsABC (Grindley lab ligase)</li>
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<img src="https://static.igem.org/mediawiki/2010/a/ae/Yale-dual-purpose-gel.jpg" />
<img src="https://static.igem.org/mediawiki/2010/a/ae/Yale-dual-purpose-gel.jpg" />
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<li> Based on gel it is clear that gel extraction is not causing loss of phsABC fragment.  The self-ligation results are less straightforward, as a dimerized phsABC should run near 7.2 kb (as it does faintly in lane 5), but the other self ligations have products that are strangely long and also very faint.  In the future will fun ligations much longer and will also test for the presence of some sort of inhibition of digestion by digesting the the phsABC fragment at a central AvaII cut site. </li>
<li> Based on gel it is clear that gel extraction is not causing loss of phsABC fragment.  The self-ligation results are less straightforward, as a dimerized phsABC should run near 7.2 kb (as it does faintly in lane 5), but the other self ligations have products that are strangely long and also very faint.  In the future will fun ligations much longer and will also test for the presence of some sort of inhibition of digestion by digesting the the phsABC fragment at a central AvaII cut site. </li>
<b>Double digest of gel-purified phsABC </b> <br/>
<b>Double digest of gel-purified phsABC </b> <br/>

Revision as of 00:12, 28 October 2010

iGEM Yale

lab notebook: week 7 (7/19-7/25)

  • Monday 7/19--Investigation of how pre-ligation processing affects ligation results
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  • Tuesday 7/20--Self-ligation of single phsABC digestions and serial digestion of ligation components
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  • Wednesday 7/21--analysis of self-ligation, tranformation of Biobrick Q04121, and set up of ligation attempt #6
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  • Thursday 7/22--
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  • Friday 7/23--
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  • Saturday 7/24--
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  • Sunday 7/25--
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