Difference between revisions of "Team:Paris Bettencourt/Notebook/VitaminA"
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<br>We migrated all the PCR products of our gBlocks on a gel: | <br>We migrated all the PCR products of our gBlocks on a gel: | ||
<br><img src="https://static.igem.org/mediawiki/2015/f/fa/Paris_Bettencourt_Gel_03-08_gblocksvA.jpg"></img> | <br><img src="https://static.igem.org/mediawiki/2015/f/fa/Paris_Bettencourt_Gel_03-08_gblocksvA.jpg"></img> | ||
− | <br>The bands for gBlocks vA-1.1, 1.2, 2 and 4 are at the expected position. The band for vA-1.1 is not very bright but still visible. There seems to be some unspecific binding for gBlock-3 as we can see two bands, one at the expected size and one smaller. We still decided to go on and try our Gibson Assembly, assuming the smaller DNA sequence wouldn't be that much of a problem as long as the right PCR product of gBlock 3 was present too. | + | <br>The bands for gBlocks vA-1.1, 1.2, 2 and 4 are at the expected position. The band for vA-1.1 is not very bright but still visible. There seems to be some unspecific binding for gBlock vA-3 as we can see two bands, one at the expected size and one smaller. We still decided to go on and try our Gibson Assembly, assuming the smaller DNA sequence wouldn't be that much of a problem as long as the right PCR product of gBlock vA-3 was present too. |
<br><br>DNA concentration was measured with a Nanodrop: | <br><br>DNA concentration was measured with a Nanodrop: | ||
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<br><b>Results</b> | <br><b>Results</b> | ||
<br>We didn’t have any colonies after the overnight culture of the cells transformed with the Gibson product. | <br>We didn’t have any colonies after the overnight culture of the cells transformed with the Gibson product. | ||
− | The E. Coli transformed with the HO-Poly-KanMX4-HO vector alone did grow very well (a lawn of bacteria on the plates with the 10^-1 and 10^-2 dilutions and more than 100 CFU on the 10^-3 plate. | + | The E. Coli transformed with the HO-Poly-KanMX4-HO vector alone did grow very well (a lawn of bacteria on the plates with the 10^-1 and 10^-2 dilutions and more than 100 CFU on the 10^-3 plate). |
<br><br><b>Interpretation</b> | <br><br><b>Interpretation</b> | ||
− | <br> What most likely happen is that the Gibson assembly failed. | + | <br> What most likely happen is that the Gibson assembly failed, which could be due to the unspecific binding during the PCR of gBlock vA-3, or to secondary structures at the extremities of our gBlocks which made the binding difficult. |
<br>Or maybe the Gibson Assembly worked, but we shouldn’t have kept the product overnight before transforming E. Coli with it. | <br>Or maybe the Gibson Assembly worked, but we shouldn’t have kept the product overnight before transforming E. Coli with it. | ||
− | Now the plan is to make all the PCR again | + | <br>Now the plan is to make all the PCR again. |
− | <h2> | + | <h2>Plan of the PCR</h2> |
<ul> | <ul> | ||
<li>gBlock 1.1 with primers o15.141 and o15.144</li> | <li>gBlock 1.1 with primers o15.141 and o15.144</li> |
Revision as of 10:29, 27 August 2015