Team:Yale/Our Project/Notebook/Week 6

From 2010.igem.org

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Wednesday 7/14--
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Wednesday 7/14--Copper Removal assay work and 5th attempt to ligate phsABC into terminator B0015
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<h4> Digest/Ligation Attempt #5 </h4>
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<i> Wetlab work for this day is also recorded on pages 67-68 of the hard copy lab notebook.</i>
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<li> Concerned that ligation components may not be thoroughly digested, so ran an all day digestion of phsABC, which containted 5 uL EcoRI buffer, 0.5 uL 100x BSA, 36.1 uL phsABC (at 27.7 ng/uL for 1 ug total), 1.8 uL EcoRI, 1.8 uL SpeI, and 4.8 uL water. </li>
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<i> Wetlab work for this day is also recorded on pages 67-68 of the hard copy lab notebook.</li>
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<li> Also concerned about SpeI activity, so ran the following diagnostic SpeI digestion of B0015 that will then be run on a gel versus the circularized plasmid: 5 uL NEB buffer 4, 0.5 uL 100x BSA, 4 uL B0015(1 ug DNA), 3.6 uL SpeI, and 36.9 uL water, let to run for 2 hours at 37˚C before heat killing at 80˚C for twenty minutes </li>
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<li>Simultaneously digested more B0015 with XbaI according to the same protocol as used on <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_5">7/8 </a> to ensure there will be enough vector. </li>
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<li> Purified the XbaI-digested B0015 with a standard <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/PCR_purify">PCR purification protocol </a>, eluting in 40 uL, and then digested all of the result with 3.6 uL EcoRI, 5 uL EcoRI buffer, 0.5 uL 100x BSA, and 0.9 uL of water, letting it run for two hours. An hour into the digestion, added 1 uL of CIP to the reaction, and after the digestion heat-killed the enzymes with 20 minutes at 80˚C. </li>
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<h4>Copper(II) Absorbance Calibration Curve Redo</h4>
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<li> Once again carefully make serial dilutions of copper sulfate in LB, creating a series of solutions running from 0.1 M to 1 uM and separated by a power of ten.  Absorbances prove to be highly nonlinear again, so research and find that copper(II) concentration cannot directly be measured directly spectrophotometrically. </li>
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<h4> Bacterial Survival in Copper Solution </h4>
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<li> Retrieve from incubator plates spotted with copper solution cultures from 6/13 copper growth assay.  Find that all cultures up to and including 4 mM copper levels survived, both in the transformed and untransformed LE392.  Also see a colony were the  pSB74 transformant in 50 mM copper was spotted, but as there is no growth at 10 mM, wonder if there was an accidental drip during spotting. </li>
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Revision as of 18:49, 27 October 2010

iGEM Yale

lab notebook: week 6 (7/12-7/18)

  • Monday 7/12--Copper growth assay of pSB74 transformants & continued ligation work
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  • Tuesday 7/13--Transformant Copper growth assay, ligation attempt #4 results, and copper removal assay prep
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  • Wednesday 7/14--Copper Removal assay work and 5th attempt to ligate phsABC into terminator B0015
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  • Thursday 7/15--
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  • Friday 7/16--
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