Difference between revisions of "Team:Michigan/Results"
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<p style="text-align:center; font-size:20px;"><font face="verdana"> Successfully deleted the alpha fragment in our Lacz plasmid, leaving only the omega fragment. The omega fragment complements the alpha fragment produced in the proximity-dependent ligation forming functional LacZ. This enzyme can break down X-Gal giving a colorimetric output. The plasmid is a very important part of our system, allowing alpha complementation and therefore producing a color visible to the naked eye. <br><br> | <p style="text-align:center; font-size:20px;"><font face="verdana"> Successfully deleted the alpha fragment in our Lacz plasmid, leaving only the omega fragment. The omega fragment complements the alpha fragment produced in the proximity-dependent ligation forming functional LacZ. This enzyme can break down X-Gal giving a colorimetric output. The plasmid is a very important part of our system, allowing alpha complementation and therefore producing a color visible to the naked eye. <br><br> | ||
| − | <a href="https://static.igem.org/mediawiki/2016/7/77/Bench_to_Bedside_MSBT_PDF.pdf" target="_blank">Click to see sequencing results.</a>font><br | + | <a href="https://static.igem.org/mediawiki/2016/7/77/Bench_to_Bedside_MSBT_PDF.pdf" target="_blank">Click to see sequencing results.</a></font><br></p> |
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Proximity-Dependent Ligation Assay (extended)</h2><br> | Proximity-Dependent Ligation Assay (extended)</h2><br> | ||
<p style="text-align:center; font-size:20px;"><font face="verdana"> We tested the proximity dependent ligation assay using a wider range of conditions, still using an agarose gel and SYBR Gold stain to visualize the DNA bands produced. In the first lane we use a 1kb plus ladder from Thermo Fisher. Lane 2 is a 30 minute reaction at room temperature without thrombin. Lane 3 is a 30 minute reaction at room temperature with thrombin added. Lanes 4 and 5 are the same as lanes 3 and 4 but run at 37 degrees C. Lane 6 is a 4 hour reaction at room temperature without thrombin. Lane 7 is a 4 hour reaction at room temperature with thrombin added. Lanes 8 and 9 are the same as lanes 6 and 7 but run at 37 degrees C. Lane 10 is a 19 hour reaction at room temperature without thrombin. Lane 11 is a 19 hour reaction at room temperature with thrombin added. Lanes 12 and 13 are the same as lanes 10 and 11 but run at 37 degrees C. The results do not seem to show any successful ligation. Interestingly, we see faint red bands of higher molecular weight in the 19 hour reactions which were run at 37 degrees C. </font><br><hr></p> | <p style="text-align:center; font-size:20px;"><font face="verdana"> We tested the proximity dependent ligation assay using a wider range of conditions, still using an agarose gel and SYBR Gold stain to visualize the DNA bands produced. In the first lane we use a 1kb plus ladder from Thermo Fisher. Lane 2 is a 30 minute reaction at room temperature without thrombin. Lane 3 is a 30 minute reaction at room temperature with thrombin added. Lanes 4 and 5 are the same as lanes 3 and 4 but run at 37 degrees C. Lane 6 is a 4 hour reaction at room temperature without thrombin. Lane 7 is a 4 hour reaction at room temperature with thrombin added. Lanes 8 and 9 are the same as lanes 6 and 7 but run at 37 degrees C. Lane 10 is a 19 hour reaction at room temperature without thrombin. Lane 11 is a 19 hour reaction at room temperature with thrombin added. Lanes 12 and 13 are the same as lanes 10 and 11 but run at 37 degrees C. The results do not seem to show any successful ligation. Interestingly, we see faint red bands of higher molecular weight in the 19 hour reactions which were run at 37 degrees C. </font><br><hr></p> | ||
| − | <img src="https://static.igem.org/mediawiki/2016/7/72/Prox-dep_assay_extended.JPG" width="500" height="500" style="float:center"> | + | <img src="https://static.igem.org/mediawiki/2016/7/72/Prox-dep_assay_extended.JPG" width="500" height="500" style="float:center"><br> |
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Revision as of 03:25, 20 October 2016
Results
Isolated LacZ from iGem part BBa_K564012
CAPTION HERE
Transferring LacZ into pET28
In order to have a control for our cell-free expression system, we cloned LacZ into the protein expression plasmid to compare LacZ expression to expression of our system with and without the presence of its target protein.
Creation of delta-M15 Mutant
Successfully deleted the alpha fragment in our Lacz plasmid, leaving only the omega fragment. The omega fragment complements the alpha fragment produced in the proximity-dependent ligation forming functional LacZ. This enzyme can break down X-Gal giving a colorimetric output. The plasmid is a very important part of our system, allowing alpha complementation and therefore producing a color visible to the naked eye.
Click to see sequencing results.
Click to see sequencing results.
