Team:Yale/Our Project/Notebook/Week 4

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<li id="nb"><a href="http://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li>
<li id="nb"><a href="http://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li>
<li id="nb"><a href="http://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li>
<li id="nb"><a href="http://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li>
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<li id="nb"><a href="http://2010.igem.org/Team:Yale/Our Project/Notebook/Week 9">week 9</a></li>
<li><a href="http://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li>
<li><a href="http://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li>
<li><a href="http://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li>
<li><a href="http://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li>
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<h4>PCR amplification of phs </h4>  
<h4>PCR amplification of phs </h4>  
<li> Because the gel run the previous day was no longer good, had to run a second 0.8% agarose gel of the PCR products to determine the success of the PCR reaction. Below is the image of the gel, with a 1 kb ladder in the leftmost lane, phsC in next lane, phsAB in lane 3, and phsABC in lane 4. </li>
<li> Because the gel run the previous day was no longer good, had to run a second 0.8% agarose gel of the PCR products to determine the success of the PCR reaction. Below is the image of the gel, with a 1 kb ladder in the leftmost lane, phsC in next lane, phsAB in lane 3, and phsABC in lane 4. </li>
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<br />
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The phsC fragment seems to have been amplified with decent success, but phsABC and phAB are only present in trace amounts, so the thermocycler protocol will have to be tweaked. <br/>
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<div align="center">
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<img src="http://2010.igem.org/wiki/images/6/68/Yale-1st-phs-pcr.jpg" />
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</div>
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<div id="caption">
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The phsC fragment seems to have been amplified with decent success, but phsABC and phAB <br /> are only present in trace amounts, so the thermocycler protocol will have to be tweaked. </div> <br />
<li> In the mean time, gel extract the phsC from lane 2 and the phsABC from lane 4 using the microcentrifuge variant of the  <a href="http://2010.igem.org/Team:Yale/Our_Project/Protocols/gel_extract">standard gel extraction protocol </a> </li>
<li> In the mean time, gel extract the phsC from lane 2 and the phsABC from lane 4 using the microcentrifuge variant of the  <a href="http://2010.igem.org/Team:Yale/Our_Project/Protocols/gel_extract">standard gel extraction protocol </a> </li>
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</ul>
</ul>
<b> Cultures for miniprep & glycerol stock redo </b> <br/>
<b> Cultures for miniprep & glycerol stock redo </b> <br/>
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In preparation for potential ligation failure, once again inoculated liquid cultures of Amp LB  for miniprep with pSB74,P0312,R0011, B0015, and J23114 transformants, making two 5 mL cultures for each plasmid.  When OD reached 0.6, once again made glycerol stocks from 0.5 mL of each solution and 0.5 mL of filter-sterilized 50% glycerol.  This time flash-froze the stocks immediately, rather than waiting overnight as on <a href="http://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_3" 6/23.</a>  <br/>
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In preparation for potential ligation failure, once again inoculated liquid cultures of Amp LB  for miniprep with pSB74,P0312,R0011, B0015, and J23114 transformants, making two 5 mL cultures for each plasmid.  When OD reached 0.6, once again made glycerol stocks from 0.5 mL of each solution and 0.5 mL of filter-sterilized 50% glycerol.  This time flash-froze the stocks immediately, rather than waiting overnight as on <a href="http://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_3">6/23</a>  <br/>
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<h4> TSI agar assay </h4>
<h4> TSI agar assay </h4>
<ul>
<ul>
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<li>LE392 and DH5alpha cultures in TSI Agar showed some growth. No black color change was visible, but both slants had a rotten egg scent, so suspected generation of H2S at concentrations too low to visibly trigger color change yet.</li><br/>
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<li>LE392 and DH5alpha cultures in TSI agar showed some growth. No black color change was visible, but both slants had a rotten egg scent, so suspected generation of H<sub>2</sub>S at concentrations too low to visibly trigger color change yet.</li><br/>
</ul>
</ul>
<h4>PCR work</h4>
<h4>PCR work</h4>
<ul>
<ul>
<li>Ran 1.0% agarose gel of four 6/30 PCR products at 90 V.</li>
<li>Ran 1.0% agarose gel of four 6/30 PCR products at 90 V.</li>
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<br />
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<div align="center">
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<img src="http://2010.igem.org/wiki/images/8/87/Yale-phs-gel.jpg" />
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</div>
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<br />
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<div id="caption">
Gel of phsABC and phsAB PCR products or a demonstration of the utility of DMSO Lane 1: 1 kb ladder and 3.6 kb spillover from Lane 2, Lane 2: 3.6 kb phsABC fragment from PCR protocol without DMSO, Lane 4: 2.9 kb phsAB fragment from PCR protocol without DMSO, Lane 6:3.6 kb phsABC fragment from PCR protocol with DMSO, Lane 8: 2.9 kb phsAB fragment from PCR protocol with DMSO
Gel of phsABC and phsAB PCR products or a demonstration of the utility of DMSO Lane 1: 1 kb ladder and 3.6 kb spillover from Lane 2, Lane 2: 3.6 kb phsABC fragment from PCR protocol without DMSO, Lane 4: 2.9 kb phsAB fragment from PCR protocol without DMSO, Lane 6:3.6 kb phsABC fragment from PCR protocol with DMSO, Lane 8: 2.9 kb phsAB fragment from PCR protocol with DMSO
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       Gel of phsABC and phsAB PCR products or a demonstration of the utility of DMSO Lane 1: 1 kb ladder and 3.6 kb spillover from Lane 2, Lane 2: 3.6 kb phsABC fragment from PCR protocol without DMSO, Lane 4: 2.9 kb phsAB fragment from PCR protocol without DMSO, Lane 6:3.6 kb phsABC fragment from PCR protocol with DMSO, Lane 8: 2.9 kb phsAB fragment from PCR protocol with DMSO <br/>
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       Gel of phsABC and phsAB PCR products or a demonstration of the utility of DMSO Lane 1: 1 kb ladder and 3.6 kb spillover from Lane 2, Lane 2: 3.6 kb phsABC fragment from PCR protocol without DMSO, Lane 4: 2.9 kb phsAB fragment from PCR protocol without DMSO, Lane 6:3.6 kb phsABC fragment from PCR protocol with DMSO, Lane 8: 2.9 kb phsAB fragment from PCR protocol with DMSO
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</div>
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<br />
<li>Based on gel, it appears that the phs50 thermocycler protocol is an improvement from phs protocol and that including DMSO also improves yield</li>
<li>Based on gel, it appears that the phs50 thermocycler protocol is an improvement from phs protocol and that including DMSO also improves yield</li>
<li>Cut out largest band for each lane and ran through <a href="http://2010.igem.org/Team:Yale/Our_Project/Protocols/gel_extract">standard gel extraction protocol.</a></li>  
<li>Cut out largest band for each lane and ran through <a href="http://2010.igem.org/Team:Yale/Our_Project/Protocols/gel_extract">standard gel extraction protocol.</a></li>  
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<b>Stockpiling plasmids for later</b> <br/>
<b>Stockpiling plasmids for later</b> <br/>
<ul>
<ul>
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<li>Miniprepped LE392 cultures grown overnight,resulting in two Eppendorfs apiece of the following plasmids: B0015 (double terminator), J23114 (constitutive promoter), pSB74 (thiosulfate reductase), R0011 (IPTG-inducible promoter), and P0312 (lacI needed with R0011). Nanodropping gave the following concentrations:</li><br/>
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<li><a href="http://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep">Miniprepped </a> LE392 cultures grown overnight,resulting in two Eppendorfs apiece of the following plasmids: B0015 (double terminator), J23114 (constitutive promoter), pSB74 (thiosulfate reductase), R0011 (IPTG-inducible promoter), and P0312 (lacI needed with R0011). Nanodropping gave the following concentrations:</li><br/>
<table>
<table>
<tr>
<tr>
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<li>
<li>
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Fridays short content
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Friday 7/2--Confirmation of hydrogen sulfide production plus more ligation work
</li>
</li>
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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> TSI Agar slants </h4>
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<ul>
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<li>Slant inoculated with the LE392 transformants containing pSB74 showed a mass of black precipitate near the surface, definitively confirming the production of hydrogen sulfide (!), the smell of which was by now very strong. The DH5alpha transformants did not exhibit the same H<sub>2</sub>S odor.</li>
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<div align="center">
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<img src="http://2010.igem.org/wiki/images/3/38/Yale-tsi-agar.png" />
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</div>
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      <div id="caption">blurry (apologies!) image of LE392 slant showing FeS precipitate(right) next to un-inoculated slant (left)</div>
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</ul>
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<h4>Ligation Attempt #2</h4>
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<ul>
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<li>Still don't know whether ligation attempt #1 was successful, as colony PCR on 7/1 was not definitive. Will sequence the results for a certain answer, but given the small number of colonies think it best to redo ligation in the interim in case of failure.</li>
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<li>To this effect redid double digestion of phsABC insert with EcoRI and SpeI and the serial digestion of B0015 with EcoRI and then XbaI. Simultaneously digested J23114 to be inserted into P0312, which lacks a promoter. Each digestion reaction had a total volume of 100 uL and included 2 ng of DNA, double the amount used previously, so that even after purification there would still be a sizable amount of DNA remaining for ligation. Otherwise digestion and purification was run the same as on 6/29 & 6/30. </li>
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</ul>
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<i>This day's activities are also recorded on pages 42-45 of the hard copy lab notebook </i>
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</div>
</div>
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<li>
<li>
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Saturday short content
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Saturday 7/3--More attempts to put a terminator on thiosulfate reductase and a promoter on lacI
</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>Ligation Attempt #2 continued</h4>
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<ul>
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<li>Following digestion and purification, took concentrations of digestion products, which were still miserably low (phsABC: 3.2 ng/uL, P0312: 16.88 ng/uL, J23114: 23.98 ng/uL, B0015: 13.24 ng/uL) and set-up ligation attempt #2. For each of the two ligations (phsABC into B0015 and J23114 into P0312) four different 20 uL reactions were run: a control ligation with vector, ligase and buffer but no insert, a reaction with a 2:1 stoichiometric ratio of insert to vector, a reaction with a 3:1 insert to vector ratio, and a reaction with a 5:1 insert to vector ratio. All reactions used 1 uL NEB T4 ligase and 2 uL of accompanying 10x buffer and were run for 15 minutes at room temperature. Following this the (theoretically) ligated plasmids were transformed into competent XL1-Blue cells according to the <a href="http://2010.igem.org/Team:Yale/Our_Project/Protocols"> standard transformation protocol </a> and plated onto Amp plates</li>
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<li>Index plates from ligation attempt #1 on 7/1 belatedly showed growth. </li>
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</ul>
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<i>This day's activities are also recorded on page 45 of the hard copy lab notebook </i>
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</div>
</div>
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<li>
<li>
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Sunday short content
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Sunday 7/4--Ligation attempt #2 transformants
</li>
</li>
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<div style="display:none;" id="sunday">
<div style="display:none;" id="sunday">
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Extra content
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<h4> Transformed Colonies from Ligation Attempt #2</h4>
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<ul>
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<li>Checked on plated transformants and observed the following: </li>
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<b>phsABC/B0015 system</b><br/>
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Reaction #1 (control) 2 colonies<br/>
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Reaction #2 (2:1 insert:vector) 4 colonies, numbered 1B-4B<br/>
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Reaction #3 (2:1 insert:vector) 5 colonies, numbered 5B-9B<br/>
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Reaction #4 (large control) 0 colonies<br/>
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<b>J23114/P0312 system </b> <br/>
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Reaction #5 (control) numerous* colonies <br/>
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Reaction #6 (2:1 insert:vector) numerous colonies, somewhat more than #5<br/>
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Reaction #7 (3:1 insert:vector) numerous colonies,about double #5<br/>
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Reaction #8 (5:1 insert:vector) numerous colonies, about double #5<br/>
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numerous=more than readily countable<br/>
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<li>Inoculated 5 mL liquid Amp/LB cultures with colonies 1B-9B and let grow overnight at 37 on shaker </li>
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<li> Set aside plates 6-8 in the fridge to work with later. </li>
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</ul>
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<i>This day's activities are also recorded on page 46 in the hard copy of the lab notebook. </i>
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</div>
</div>

Latest revision as of 02:50, 28 October 2010

iGEM Yale

lab notebook: week 4 (6/28 -7/4)

  • Monday 6/28--PCR amplification of thiosulfate reductase operon phsABC
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  • Tuesday 6/29--Gel extraction of PCR amplified phs, TSI agar slant assay for hydrogen sulfide production, & pre-ligation double digestion
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  • Wednesday 6/30--Troubleshooting PCR amplification of thiosulfate reductase gene & continued work toward ligation
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  • Thursday 7/1--Smelly bacteria (yay!) & ongoing ligation efforts
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  • Friday 7/2--Confirmation of hydrogen sulfide production plus more ligation work
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  • Saturday 7/3--More attempts to put a terminator on thiosulfate reductase and a promoter on lacI
  • See more/less
  • Sunday 7/4--Ligation attempt #2 transformants
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