Team:Yale/Our Project/Notebook/Week 7

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<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></b></li>
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></b></li>
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li>
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li>
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<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 9">week 9</a></li>
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li>
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li>
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li>
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li>
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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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<br />
<div align="center">
<div align="center">
<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" />
</div>
</div>
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<br />
<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/>
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<ul>
<ul>
<li>Ran a 1.0% agarose gel with the six self-ligation reactions.  They were run at 90 V against a 1 kb ladder and pSB74.</li>
<li>Ran a 1.0% agarose gel with the six self-ligation reactions.  They were run at 90 V against a 1 kb ladder and pSB74.</li>
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<br />
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<div align="center">
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<img src="https://static.igem.org/mediawiki/2010/a/a9/Yale-self-ligation.jpg" />
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</div>
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<br />
From left to right the lanes are: Lane 1--1 kb ladder, Lane 2--circular pSB74 (about 8 kb), Lane 3--ligation of SpeI digest of gel extracted phsABC, Lane 4--Lane 3--ligation of SpeI digest of PCR clean-up phsABC, Lane 5--ligation of AvaII digest of gel extracted phsABC, Lane 6--ligation of AvaII digest of PCR cleanup phsABC, Lane 7--ligation of EcoRI digest of gel extracted phsABC, Lane 8--ligation of EcoRI digest of PCR clean-up phsABC <br/>
From left to right the lanes are: Lane 1--1 kb ladder, Lane 2--circular pSB74 (about 8 kb), Lane 3--ligation of SpeI digest of gel extracted phsABC, Lane 4--Lane 3--ligation of SpeI digest of PCR clean-up phsABC, Lane 5--ligation of AvaII digest of gel extracted phsABC, Lane 6--ligation of AvaII digest of PCR cleanup phsABC, Lane 7--ligation of EcoRI digest of gel extracted phsABC, Lane 8--ligation of EcoRI digest of PCR clean-up phsABC <br/>
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<li>The AvaII bands are too faint to see really, but the SpeI and EcoRI ligations appear to show bands near 7.3, where dimers would show if they formed, suggesting that the longer ligation time may have helped.  In general though, the gel never resolved well even when the loading dye was run off the end. </li>
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<li>The AvaII bands are too faint to see really, but the SpeI and EcoRI ligations appear to show bands near 7.3 kb, where dimers would show if they formed, suggesting that the longer ligation time may have helped.  In general though, the gel never resolved well even when the loading dye was run off the end. </li>
</ul>
</ul>
<h4>Ligation using serial digestion products </h4>
<h4>Ligation using serial digestion products </h4>
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<b>Quick ligase control ligation</b>--10 uL ligase buffer, 1 uL Quick ligase, 3.5 uL B0015, 6.5 uL water <br/>
<b>Quick ligase control ligation</b>--10 uL ligase buffer, 1 uL Quick ligase, 3.5 uL B0015, 6.5 uL water <br/>
<b>Quick ligase 2:1 insert:vector</b>--10 uL ligase buffer, 1 uL Quick ligase, 3.5 uL B0015, 6.5 uL phsABC <br/>
<b>Quick ligase 2:1 insert:vector</b>--10 uL ligase buffer, 1 uL Quick ligase, 3.5 uL B0015, 6.5 uL phsABC <br/>
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<i>This day's labwork is also recorded on pages 76-78 of the hard copy lab notebook </i>
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<li> Also did ligations of nonsequentially digested DNA to see if it would work, following the plan outlined below. For the phsABC tried both the gel extraction product and the PCR clean-up product, while with B0015 stuck to the not CIP-treated variety. All reactions were done using normal T4 ligase. </li>
<li> Also did ligations of nonsequentially digested DNA to see if it would work, following the plan outlined below. For the phsABC tried both the gel extraction product and the PCR clean-up product, while with B0015 stuck to the not CIP-treated variety. All reactions were done using normal T4 ligase. </li>
<b>control ligation </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 14.5 uL water <br/>
<b>control ligation </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 14.5 uL water <br/>
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<li>
<li>
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Thursday 7/22--
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Thursday 7/22--Ligation Transformations
</li>
</li>
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Extra content
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<b> Q02141 transformation </b> <br/>
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<ul>
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<li>
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No growth was observed on the Q04121 plates--may need to find better competent cells. </li>
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</ul>
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<h4> Transformation of Varied Ligation Efforts </h4>
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<ul>
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<li> Retrieved ligations from overnight water bath and heat-killed the T4 ligase with ten minutes at 65˚C. </li>
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<li> After previous ligation troubles, have invested in commercial grade ultracompetent cells.  Transformed all of the 7/21 ligations into Top10 One Shot cells according to the standard <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation"> transformation protocol </a>, also transforming the pUC19 standard that came with the cells.  All of these were plated on ampicillin plates, but also transformed DH5alpha cells with Q04121 and plated that with kanamycin. </li>
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</ul>
<i>This day's labwork is also recorded on page 79 of the hard copy lab notebook </i>
<i>This day's labwork is also recorded on page 79 of the hard copy lab notebook </i>
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<li>
<li>
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Friday 7/23--
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Friday 7/23--Results of ligation transformations
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<div style="display:none;" id="friday">
<div style="display:none;" id="friday">
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Extra content
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<h4>Ligation Transformants </h4>
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<ul>
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<li> The plated Q04121 had two colonies, while the ethanol precipitated DNA ligation control, Quick ligase control, and the ethanol precipitated 2:1 plates all had more colonies than readily countable.  The Quick ligase 2:1 had noticeably fewer, and the ethanol precipitated 6:1 ligation gave no colonies at all.  In terms of the other transformations, the pUC19, non-ethanol precipitated control, PCR clean-up 6:1 ligation, PCR cleanup 2:1, ligation, and gel extracted 2:1 ligation had lots of colonies, with somewhat fewer on the gel extracted 6:1 ligation plate. </li>
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<li> Chose four colonies from each of the noncontrol ligations and performed colony PCR, lysing the colonies in water before and  mixing  a uL of the resulting solution with the following: 2 uL VF2 primer (10 mM), 2 uL VR primer (10 mM), 10.8 uL water, 4 uL of Phusion buffer, 0.6 uL of DMSO, 0.4 uL dNTPs, and 0.2 uL of Phusion polymerase.  Ran the resulting PCR reaction on the "VR" protocol of the thermocycler. </li>
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<li> Also started liquid cultures of each of the colonies in ampicillin LB. </li>
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<li> The colonies were labeled as follows: The four from the Quick Ligase ligation were labeled #1-#4, those from the PCR cleanup 2:1 ligation sere labeled #5-#8, those from the 2:1 ethanol precipitated DNA ligation were called #9-#12, those form the gel extracted 6:1 ligation were #13-#16, while those from the gel extracted 2:1 ligation were numbered #17-#20, and finally those from the PCR clean-up 6:1 ligation were labeled #21-#24 </li>
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</ul>
<i>This day's labwork is also recorded on pages 79 & 80 of the hard copy lab notebook </i>
<i>This day's labwork is also recorded on pages 79 & 80 of the hard copy lab notebook </i>
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<li>
<li>
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Saturday 7/24--
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Saturday 7/24--Gel of colony PCR suggest a successful ligation of phsABC into B0015!
</li>
</li>
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<div style="display:none;" id="saturday">
<div style="display:none;" id="saturday">
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Extra content
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<h4> Gel of Colony PCR </h4>
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<ul>
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<li> Ran the twenty-four colony PCR reactions from 7/23 on a single massive 1.0% agarose gel vs a 1 kb ladder at 90 V.</li>
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<div align="center">
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<img src="https://static.igem.org/mediawiki/2010/f/f6/Yale-colony-pcr.jpg" />
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</div>
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<div id="caption">
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Ladder is very faint in the leftmost lane and the twenty-four reactions are in order from left to right.  Despite the faintness, one can see that samples 5, 12, 14, 15, 20, 21 and 24 appear to have the desired 3.6 kb fragment indication a successful ligation.</div>
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</ul>
<i>This day's labwork is also recorded on page 81 of the hard copy lab notebook </i>
<i>This day's labwork is also recorded on page 81 of the hard copy lab notebook </i>
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<li>
<li>
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Sunday 7/25--
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Sunday 7/25--Inoculate cultures of likely ligations successes for miniprep on 7/26
</li>
</li>
<a id="link" href="javascript:ReverseDisplay('sunday')">See more/less</a>
<a id="link" href="javascript:ReverseDisplay('sunday')">See more/less</a>
<div style="display:none;" id="sunday">
<div style="display:none;" id="sunday">
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<b> Inoculation of liquid cultures </b> <br/>
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<ul>
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<li> Inoculated liquid ampicillin in LB cultures from the colonies shown to contain successful ligations by the 9/25 gel of colony PCR.  Also inoculated a culture of Q04121 for overnight growth, adding kanamycin to a concentration of 50 ng/uL. </li>
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</ul>
<i>This day's labwork is also recorded on page 81 of the hard copy lab notebook </i>
<i>This day's labwork is also recorded on page 81 of the hard copy lab notebook </i>
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Latest revision as of 02:53, 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--Ligation Transformations
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  • Friday 7/23--Results of ligation transformations
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  • Saturday 7/24--Gel of colony PCR suggest a successful ligation of phsABC into B0015!
  • See more/less
  • Sunday 7/25--Inoculate cultures of likely ligations successes for miniprep on 7/26
  • See more/less