Difference between revisions of "Part:BBa K2429050:Experience"

 
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===Applications of BBa_K2429050===
 
===Applications of BBa_K2429050===
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<h3> Verifying the construct </h3>
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<h4> mKate Titration </h4>
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<p>Before being able to test our guides against our newly designed reporter, we needed to ensure it was exhibiting proper behavior. To that end, we ran an titration on the mKate-HBG construct. We expected that as we increased the amount of reporter in the system, the amount of mKate would rise, as the reporter was designed to output normally in the absence of our system. These expected results can be seen below. The different colored lines corrispond to different <a href= "http://2017.igem.org/Team:MIT/HowtoreadaCytoflowgraph"> transfection bins</a> </p>
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<center><img src="https://static.igem.org/mediawiki/2017/5/5c/Expected_output_of_mKate_titration.png" alt="Expected output" style= "width:50%;"></center>
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<p> In our experiment, we varied the amounts of mKate-HBG from 10 to 500 ng. (For a detailed explanation of how to plan a mammalian transfection <a href= "https://static.igem.org/mediawiki/2017/6/63/Planning_Mamallian_Transfections_%C2%B7_Benchling.pdf"> click here</a>) </p>
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<center><h4>mKate Titration for mKate-HBG</h4></center>
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<center><img src="https://static.igem.org/mediawiki/2017/1/15/MkateHBG_noRBP.svg" alt="Figure ASO3vsmkateamt_red" style= "width:50%;"></center>
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<p><i>mKate-HBG reporter amounts (10ng-500ng) vs. the amount of red fluorescence (AU). The color of the line indicate the <a href= "http://2017.igem.org/Team:MIT/HowtoreadaCytoflowgraph"> transfection bins</a> of each result.</i></p>
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<p>The reporter is not producing red fluorescence above background noise. </p>
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<strong>Unfortunately, we were unable to test any of our constructs against this reporter, because the reporter was not producing red.</strong> '
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<p>Our next step is to debug our reporter. We suspect the problem may be with the intron we chose, or the way we built that intron, because the mKate-ff4 was outputting fine.</p>
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===User Reviews===
 
===User Reviews===

Latest revision as of 18:30, 1 November 2017


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K2429050

Verifying the construct

mKate Titration

Before being able to test our guides against our newly designed reporter, we needed to ensure it was exhibiting proper behavior. To that end, we ran an titration on the mKate-HBG construct. We expected that as we increased the amount of reporter in the system, the amount of mKate would rise, as the reporter was designed to output normally in the absence of our system. These expected results can be seen below. The different colored lines corrispond to different transfection bins

Expected output

In our experiment, we varied the amounts of mKate-HBG from 10 to 500 ng. (For a detailed explanation of how to plan a mammalian transfection click here)

mKate Titration for mKate-HBG

Figure ASO3vsmkateamt_red

mKate-HBG reporter amounts (10ng-500ng) vs. the amount of red fluorescence (AU). The color of the line indicate the transfection bins of each result.

The reporter is not producing red fluorescence above background noise.

Unfortunately, we were unable to test any of our constructs against this reporter, because the reporter was not producing red. '

Our next step is to debug our reporter. We suspect the problem may be with the intron we chose, or the way we built that intron, because the mKate-ff4 was outputting fine.

User Reviews

UNIQ5f276b304af7b2ed-partinfo-00000001-QINU UNIQ5f276b304af7b2ed-partinfo-00000002-QINU