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| <p style="font-size:17px;" align="justify"></br><h7 style="color:purple"><b>Nanodrop Analysis</b></h7> | | <p style="font-size:17px;" align="justify"></br><h7 style="color:purple"><b>Nanodrop Analysis</b></h7> |
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− | <br/> We amplified different EC DNA by PCR and ran a gel electrophoresis to analyze the results. | + | <br/> We used the Nanodrop to analyze the DNA extracted with a microneedle patch (red). The positive control (green) is EC synthetic DNA sequence and the negative control (blue) is the elution buffer applied on an unused microneedle patch. </br> |
− | We loaded the gel from left to right as following, the ladder, synthetic EC DNA sequence,
| + | As expected, the negative control absorption at 260nm is close to zero, no DNA is present in the microneedle patch. In contrast, the positive control has the typical DNA Nanodrop readout. </br> |
− | DNA extracted with a microneedle patch, DNA extracted with a standard kit, control with primers but no DNA.
| + | The graph of DNA extracted by microneedle patch shows a peak of absorption at 260nm, we can also see that that values absorption values at 230nm and 280nm are lower that the value at 260nm. Overall, the graph is similar to the positive control but with lower values. This indicates that DNA was extracted by the microneedle patch. |
− | Bands are observed in all the lanes containing DNA, no band is present in the control. This shows that the
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− | microneedle patch successfully extracted DNA from the plant, which was then amplified by PCR.
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| </p> | | </p> |
| <img src="https://static.igem.org/mediawiki/2019/2/22/T--EPFL--courbes.png" > | | <img src="https://static.igem.org/mediawiki/2019/2/22/T--EPFL--courbes.png" > |
− | <p style="font-size:17px;" align="justify">
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− | <br/> We used the Nanodrop to analyze the DNA extracted with a microneedle patch (red).
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− | The positive control (green) is EC synthetic DNA sequence and the negative control (blue) is the elution buffer applied on an unused microneedle patch. </br>
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− | As expected, the negative control absorption at 260nm is close to zero, no DNA is present in the microneedle patch.
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− | In contrast, the positive control has the typical DNA Nanodrop readout. </br>
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− | The graph of DNA extracted by microneedle patch shows a peak of absorption at 260nm, we can also see that that
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− | values absorption values at 230nm and 280nm are lower that the value at 260nm. Overall, the graph is similar to the
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− | positive control but with lower values. This indicates that DNA was extracted by the microneedle patch.
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− |
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− | </p>
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| </div> | | </div> |
| </div> | | </div> |
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| </p> | | </p> |
| + | </br> |
| + | </br> |
| + | <p style="font-size:17px; align="justify"> |
| + | <h7 style="color:purple"><b>Transcription </b></h7> |
| + | </br> |
| + | </br> |
| + | To test that our amplicons could indeed be transcribed, we did an <i>in vitro</i> transcription reaction using HiScribe™ T7 Quick High Yield RNA Synthesis Kit. The transcription product were detected by electrophoresis on a 2% denaturing agarose gel. |
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| + | </p> |
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| + | <img src="https://static.igem.org/mediawiki/2019/9/9a/T--EPFL--transcription_rpa.jpg" height=auto width=auto > |
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| + | <p style="font-size:13px"><i> Figure 12 : Agarose gel electrophoresis of post-RPA transcription product</i></p> |
| + | <p style="font-size:17px; align="justify"> |
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| + | The transcription of all 3 RPA products (lanes 1, 4 and 7) is similar to that or their synthetic counterpart sequences with T7 (lanes 2, 5 and 8). This means that RPA was successful in adding a functional T7 promoter to the amplified sequences. |
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| + | </p> |
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| </div> | | </div> |