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Team:CSU Fort Collins/Notebook/Breakdown/Sep
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| - | <li class='last'><a href='/Team:CSU_Fort_Collins/ | + | <li><a href='/Team:CSU_Fort_Collins/Sponsors/'><span>Sponsors</span></a></li> |
| + | <li class='last'><a href='/Team:CSU_Fort_Collins/Acknowledgements/'><span>Acknowledgements</span></a></li> | ||
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| - | <li class='has-sub'><a href='/Team:CSU_Fort_Collins/Notebook/Breakdown/'><span>Breakdown</span></a> | + | <li class='has-sub'><a href='/Team:CSU_Fort_Collins/Notebook/Breakdown/Jul'><span>Breakdown</span></a> |
<ul> | <ul> | ||
| - | <li><a href=" | + | <li><a href="/Team:CSU_Fort_Collins/Notebook/Breakdown/Jul"><span>July</span></a></li> |
| - | <li><a href=' | + | <li><a href='/Team:CSU_Fort_Collins/Notebook/Breakdown/Aug'><span>August</span></a></li> |
| - | <li class='last'><a href=" | + | <li class='last'><a href="/Team:CSU_Fort_Collins/Notebook/Breakdown/Sep"><span>September</span></a></li> |
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| - | <li class='has-sub'><a href='/Team:CSU_Fort_Collins/Notebook/HVP/'><span>High-Value Product</span></a> | + | <li class='has-sub'><a href='/Team:CSU_Fort_Collins/Notebook/HVP/Jun'><span>High-Value Product</span></a> |
<ul> | <ul> | ||
| - | <li><a href=" | + | <li><a href="/Team:CSU_Fort_Collins/Notebook/HVP/Jun"><span>June</span></a></li> |
| - | + | <li class='last'><a href="/Team:CSU_Fort_Collins/Notebook/HVP/Jul"><span>July</span></a></li> | |
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| - | <li class='last'><a href=" | + | |
</ul> | </ul> | ||
</li> | </li> | ||
| - | <li li class='has-sub'><a href='/Team:CSU_Fort_Collins/Notebook/KillSwitch/'><span>Kill Switch</span></a> | + | <li li class='has-sub'><a href='/Team:CSU_Fort_Collins/Notebook/KillSwitch/Jul'><span>Kill Switch</span></a> |
<ul> | <ul> | ||
| - | <li><a href=" | + | <li><a href="/Team:CSU_Fort_Collins/Notebook/KillSwitch/Jul"><span>July</span></a></li> |
| - | + | <li class='last'><a href="/Team:CSU_Fort_Collins/Notebook/KillSwitch/Sep"><span>September</span></a></li> | |
| - | <li class='last'><a href=" | + | |
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| - | <li class='has-sub'><a href='/Team:CSU_Fort_Collins/ | + | <li class='has-sub'><a href='/Team:CSU_Fort_Collins/Collab/'><span>Human Practices</span></a> |
<ul> | <ul> | ||
<li><a href='/Team:CSU_Fort_Collins/Collab/'><span>Collaboration</span></a></li> | <li><a href='/Team:CSU_Fort_Collins/Collab/'><span>Collaboration</span></a></li> | ||
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| - | <li class='has-sub'><a href='/Team:CSU_Fort_Collins/ | + | <li class='has-sub'><a href='/Team:CSU_Fort_Collins/Parts/'><span>Achievements</span></a> |
<ul> | <ul> | ||
<li><a href='/Team:CSU_Fort_Collins/Parts/'><span>Parts</span></a></li> | <li><a href='/Team:CSU_Fort_Collins/Parts/'><span>Parts</span></a></li> | ||
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| - | <li><a href='/Team:CSU_Fort_Collins/Safety/'><span>Safety</span></a></li> | + | <li class='last'><a href='/Team:CSU_Fort_Collins/Safety/'><span>Safety</span></a></li> |
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<div class="month-group"> | <div class="month-group"> | ||
<a href='/Team:CSU_Fort_Collins/Notebook/Breakdown/Jul'>July</a> | <a href='/Team:CSU_Fort_Collins/Notebook/Breakdown/Jul'>July</a> | ||
| + | </div> | ||
<p>Lac Promoter and FadD Assembly</p> | <p>Lac Promoter and FadD Assembly</p> | ||
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<div class="month-group"> | <div class="month-group"> | ||
<a href='/Team:CSU_Fort_Collins/Notebook/Breakdown/Aug'>August</a> | <a href='/Team:CSU_Fort_Collins/Notebook/Breakdown/Aug'>August</a> | ||
| + | </div> | ||
<p>More of the Same, But Better</p> | <p>More of the Same, But Better</p> | ||
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<div class="month-group"> | <div class="month-group"> | ||
<a href='/Team:CSU_Fort_Collins/Notebook/Breakdown/Sep'>September</a> | <a href='/Team:CSU_Fort_Collins/Notebook/Breakdown/Sep'>September</a> | ||
| + | </div> | ||
<p>One Thousand and One Nights</p> | <p>One Thousand and One Nights</p> | ||
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<h4>Thursday, September 4</h4> | <h4>Thursday, September 4</h4> | ||
| - | Choose new lac promoter, part BBa_J04500, from 2014 Distribution Kit, plate 3 well 19J. Transformed part into E. coli. | + | Choose new lac promoter, part BBa_J04500, from 2014 Distribution Kit, plate 3 well 19J. Transformed part into <u>E. coli</u>. |
<h4>Friday, September 5</h4> | <h4>Friday, September 5</h4> | ||
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<h4>Tuesday, October 7</h4> | <h4>Tuesday, October 7</h4> | ||
| - | Transformed final plasmid into E. coli. | + | Transformed final plasmid into <u>E. coli</u>. |
<h4>Wednesday, October 8</h4> | <h4>Wednesday, October 8</h4> | ||
Latest revision as of 20:16, 17 October 2014
Breakdown Daily Notes
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Lac Promoter and FadD Assembly
More of the Same, But Better
One Thousand and One Nights
SEPTEMBER
Monday, September 1
Miniprepped the overnight cultures and analyzed with nanodrop to find the concentrations.Tuesday, September 2
Talked to our advisor Dr. Peebles about why the part had not assembled correctly. Discussed that our primers for FadD should have Xba1, Spe1 and Pst1 cut sites. Ran a restriction enzyme digest with EcoR1 and Pst1 to remove the insert and make the plasmid linearized. Also cut the lac promoter plasmid with EcoR1 and Pst1 for the same reason. Ran a gel to determine if the plasmids were correctly assembled. The gel failed.Wednesday, September 3
Discussed primers and gels with our advisor Dr. Peebles. Discovered that our primers should have a Xba1 cut site not an Xho1 site. Redesigned and reordered primers. Also discovered that the ladder we were using for gels was optimized for TBE not TAE. Got a new ladder that works in TAE.Thursday, September 4
Choose new lac promoter, part BBa_J04500, from 2014 Distribution Kit, plate 3 well 19J. Transformed part into E. coli.Friday, September 5
Made culture tubes of and plated lac promoter transformation.Saturday, September 6
Minipreped the lac promoter. Took concentrations of the miniprep using a nanodrop. Ran a restriction enzyme digest on the lac promoter using Xba1 and Pst1. Ran a gel to check the lac promoter, the gel failed.Sunday, September 7
Made new culture tubes so we could miniprep the lac promoter again.Thursday, September 11
Ran a PCR to extract FadD with the new primers.Wednesday, September 17
Ran a gel to check the PCR, the gel failed. Ran a nanodrop to find the concentrations.Thursday, September 18
Transformed lac promoter from 2014 and 2013 (plate 3 well 20J) Distribution Kits. Retried the gel using SYBR green in the gel not in the well. The gel failed.Saturday, September 20
Miniprepped both lac promoters.Monday, September 22
Did a colony PCR using colonies from the lac promoter transformation. Found the concentration of the miniprepped lac promoters. Ran a restriction enzyme digest on the lac promoter plasmid using Xba1 and Pst1 so it was linear for the gel.Tuesday, September 23
Ran a gel on the PCR product and the digested lac promoter. The gel appeared to fail. The backbone was visible but the actual lac promoter was not.
Gel Results, September 23
Tuesday, September 30
Ran a PCR with protocol and primers from our PI to determine if our PCR enzymes were still good. Ran a gel with this PCR product and the same digested lac promoter. Used Dr. Peebles’ gel viewer and saw that while the lac promoter was correct, the PCR got nothing. We thus determined that our PCR polymerase was likely faulty.OCTOBER
Wednesday, October 1
Ran a PCR with our PI’s PCR machine and enzymes. Did both a control with her primers and the FadD.| PCR Thermalcycler Program | ||
| Number of cycles | Temperature (° C) | Length of time |
| 1 | 98 | 5 min |
| 5 | 98 | 30 sec |
| 5 | 57 | 25 sec |
| 5 | 72 | 2 min |
| 25 | 98 | 20 sec |
| 25 | 72 | 2 min |
| 1 | 72 | 10 min |
| 1 | 4 | - |
Friday, October 3
Ran a gel of the PCR products. The first column is the control and the second two are the FadD PCR product.
Gel Results, October 3
