|
|
(25 intermediate revisions by 2 users not shown) |
Line 55: |
Line 55: |
| <a class="dropdown-item" href="#PCR" style="font-size:17px">PCR Amplification</a> | | <a class="dropdown-item" href="#PCR" style="font-size:17px">PCR Amplification</a> |
| </li> | | </li> |
− | </br><a class="nav-link" href="#Results" style="font-size:17px" > Results </a> | + | </br> |
| + | <li class="nav-item dropdown"> |
| + | <a class="nav-link dropdown-toggle" href="#Results" role="button" aria-haspopup="true" aria-expanded="false" style="font-size:17px" > Results </a> |
| + | <a class="dropdown-item" href="#spectrophotometer" style="font-size:17px">Nanodrop analysis</a> |
| + | <a class="dropdown-item" href="#Gel" style="font-size:17px">PCR ampplification</a> |
| + | </li> |
| + | <a class="nav-link" href="#Conclusion" style="font-size:17px" > Conclusion </a> |
| <a class="nav-link" href="#References" style="font-size:17px"> References </a> | | <a class="nav-link" href="#References" style="font-size:17px"> References </a> |
| </div> | | </div> |
Line 121: |
Line 127: |
| <p style="font-size:17px; align="justify"> | | <p style="font-size:17px; align="justify"> |
| All MN patches used for DNA extraction were fabricated using polydimethylsiloxane molds. These molds were fabricated by laser ablation, and the dimension of each mold is approximately 10 mm × 10 mm, which has 15 × 15 arrays of microneedle conical cavities. The height of each cavity is 800 μm, and the diameters of the tip and base are 10 and 300 μm, respectively. To fabricate the microneedle patches, 0.5 mL of poly(vinyl alcohol) (30− 70 kDa, 10 wt %) solution was added to each silicone mold. After that, the molds are placed in a centrifuge for 20 min at 40°C at 4000 rpm to draw the PVA solution into the cavities and achieve the desired viscosity. These molds were then kept overnight at 25°C in a chemical hood vacuum. After being dried, the microneedle patches were carefully separated from the molds and stored at 25°C in a sealed Petri dish. The patches ought to be used within a month after their fabrication date.</p> | | All MN patches used for DNA extraction were fabricated using polydimethylsiloxane molds. These molds were fabricated by laser ablation, and the dimension of each mold is approximately 10 mm × 10 mm, which has 15 × 15 arrays of microneedle conical cavities. The height of each cavity is 800 μm, and the diameters of the tip and base are 10 and 300 μm, respectively. To fabricate the microneedle patches, 0.5 mL of poly(vinyl alcohol) (30− 70 kDa, 10 wt %) solution was added to each silicone mold. After that, the molds are placed in a centrifuge for 20 min at 40°C at 4000 rpm to draw the PVA solution into the cavities and achieve the desired viscosity. These molds were then kept overnight at 25°C in a chemical hood vacuum. After being dried, the microneedle patches were carefully separated from the molds and stored at 25°C in a sealed Petri dish. The patches ought to be used within a month after their fabrication date.</p> |
| + | <img src="https://static.igem.org/mediawiki/2019/f/ff/T--EPFL--ourMN.png" > |
| + | <p style="font-size:17px; align="justify">FIGURE - A picture of one of our Microneedle patches.</p> |
| <h5 style="color:indigo" id="MN">MN Patch-Based DNA Extraction</h5> <p style="font-size:17px; align="justify"> | | <h5 style="color:indigo" id="MN">MN Patch-Based DNA Extraction</h5> <p style="font-size:17px; align="justify"> |
| There were two simple steps for MN patch-based DNA extraction from a fresh plant leaf. First, a fresh MN patch is gently placed on the surface of the leaf of interest. Then, a puncture force is delivered to the patch by finger pressing for a few seconds. Finally, the patch is peeled off and rinsed with 100 μL of TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0). For each extraction, a new MN patch was used. MN- extracted solutions were used each time without further purification. Please refer to the previous video for an illustrated procedure.</p> | | There were two simple steps for MN patch-based DNA extraction from a fresh plant leaf. First, a fresh MN patch is gently placed on the surface of the leaf of interest. Then, a puncture force is delivered to the patch by finger pressing for a few seconds. Finally, the patch is peeled off and rinsed with 100 μL of TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0). For each extraction, a new MN patch was used. MN- extracted solutions were used each time without further purification. Please refer to the previous video for an illustrated procedure.</p> |
| | | |
| + | <img src="https://static.igem.org/mediawiki/2019/1/16/T--EPFL--ourMNleaf.png" > |
| + | |
| + | <p style="font-size:17px; align="justify">FIGURE - A picture of a leaf after application of a Microneedle patch. The extraction was performed three weeks before (left); one week before (right).</p> |
| | | |
| <h5 style="color:indigo" id="DNA"> DNA Extraction with the DNeasy Plant Pro Kit</h5> <p style="font-size:17px; align="justify"> | | <h5 style="color:indigo" id="DNA"> DNA Extraction with the DNeasy Plant Pro Kit</h5> <p style="font-size:17px; align="justify"> |
Line 142: |
Line 153: |
| </div> | | </div> |
| <div class="post-content"><p style="font-size:17px; align="justify"> | | <div class="post-content"><p style="font-size:17px; align="justify"> |
− | The entire process of the DNA extraction using the MN patches takes less than 1 min, orders of magnitude faster and simpler than the conventional extraction protocol.</p> </div>
| + | We compared our DNA extraction method using microneedles with the conventional extraction method, by characterizing |
− | <h5 style="color:indigo">Comparison of extraction efficiency between the MN and the Dneasy methods. </h5> | + | the quantity and purity of DNA extracted with both methods.</p> </div> |
| + | |
| + | <!-- |
| <div class="post-content"><p style="font-size:17px; align="justify"> | | <div class="post-content"><p style="font-size:17px; align="justify"> |
− | We compared our DNA extraction method using microneedles with the conventional extraction method, by characterizing the quantity and purity of DNA extracted with both methods. The total extracted DNA and the purity of DNA obtained by both extraction methods were characterized by a Nano- Drop spectrophotometer. From the spectral data, it is clear that the MN patch extracted a significant amount of DNA from plant leaves based on the appearance of Axxx absorption for all samples tested. In contrast, the solution from the blank MN patch did not show any significant absorption at xxx nm. The purity of MN-extracted DNA samples was compared with those obtained by conventional extraction method. <br> We can also note that the conventional method takes 1 to 2 hours and requires a lab, and is therefore more than 100 times more time consuming than MN extraction, which is non neglectable. </p> | + | We compared our DNA extraction method using microneedles with the conventional extraction method, by characterizing |
| + | the quantity and purity of DNA extracted with both methods. The total extracted DNA and the purity of DNA obtained by |
| + | both extraction methods were characterized by a Nano- Drop spectrophotometer. From the spectral data, it is clear |
| + | that the MN patch extracted a significant amount of DNA from plant leaves based on the appearance of A260 absorption |
| + | for all samples tested. In contrast, the solution from the blank MN patch did not show any significant absorption at |
| + | 260 nm. The purity of MN-extracted DNA samples was compared with those obtained by conventional extraction method. <br> |
| + | We can also note that the conventional method takes 1 to 2 hours and requires a lab, and is therefore more than 100 times |
| + | more time consuming than MN extraction, which is non neglectable. </p></div> |
| + | --> |
| | | |
− | | + | |
− | </div> | + | |
| | | |
| | | |
Line 155: |
Line 175: |
| <article class="post"> | | <article class="post"> |
| <div class="post-wrapper"> | | <div class="post-wrapper"> |
− | <div class="post-header">
| + | |
− | <h1 class="post-title" style="color:purple">Successful DNA Extraction</h1>
| + | <h5 style="color:indigo" id="spectrophotometer">Nanodrop analysis </h5> |
| + | |
| + | |
| + | |
| | | |
− | </div>
| |
− | <div class="post-content"><p style="font-size:17px;" align="justify">
| |
| | | |
| | | |
− | <img src="https://static.igem.org/mediawiki/2019/d/de/T--EPFL--PCR_MN_EC.jpg" >
| |
− | </div>
| |
| | | |
| | | |
− | <div class="row">
| |
− | <div class="col-md-6">
| |
− | <div class="sticky-sidebar">
| |
| | | |
− | <div class="info-list">
| + | |
| | | |
− | <p style="font-size:17px; align="justify">On the left Image (a), we are amplifying EC from (left to right): a gBlock, DNA extracted using our MicroNeedle patches, DNA extracted using a standard kit, and finally a control amplification using the corresponding primers but no DNA. The kit we used is DNeasy Plant Pro Kit by Qiagen.We can conclude that our method successfully extracts DNA from plant leaves. On the second image, we performed Nanodrop UV absorption spectra : Red Line corresponds to gBlock; Black Line corresponds to MN extraction. The DNA concentration is high enough to have an apparent signal on the graphs (bump on the curve).
| + | <div class="post-content"><p style="font-size:17px;" align="justify"> |
| + | 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> |
| + | 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> |
| + | 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. |
| + | |
| + | |
| + | <img src="https://static.igem.org/mediawiki/2019/2/22/T--EPFL--courbes.png" > |
| + | </br> </p> |
| + | <h5 style="color:indigo" id="Gel">PCR amplification</h5> |
| + | <p style="font-size:17px; align="justify"> |
| + | We amplified different EC DNA by PCR and ran a gel electrophoresis to analyze the results. |
| + | Bands are observed in all the lanes containing DNA, no band is present in the control. |
| + | This shows that the microneedle patch successfully extracted DNA from the plant, which was then amplified by PCR. |
| </p> | | </p> |
| | | |
− | </div>
| + | <img src="https://static.igem.org/mediawiki/2019/d/de/T--EPFL--PCR_MN_EC.jpg" > |
| | | |
− | </div> </div>
| |
− | <div class="col-md-6"><img src="https://static.igem.org/mediawiki/2019/3/35/T--EPFL--2courbes2.png" alt="">
| |
− | <img src="https://static.igem.org/mediawiki/2019/c/c9/T--EPFL--3courbes.png" >
| |
− | </div>
| |
| | | |
| | | |
Line 187: |
Line 211: |
| </article> | | </article> |
| | | |
| + | <article class="post"> |
| + | <div class="post-wrapper"> |
| + | <div class="post-header" > |
| + | <h2 style="color:purple" id="Conclusion">Conclusion</h2> |
| + | </div> |
| + | <div class="post-content"><p style="font-size:17px; align="justify"> |
| + | We demonstrated that microneedle patches can be used to extract DNA from leaves in less than 1 minute. |
| + | The extracted DNA could be then amplified by PCR and analyzed by gel electrophoresis. |
| + | We can also note that the conventional method takes 1 to 2 hours and requires a lab and is |
| + | therefore more than 100 times more time consuming than microneedle extraction, which is non neglectable. |
| + | |
| | | |
| + | |
| + | </div> |
| + | |
| + | |
| + | </div> |
| + | </article> |
| | | |
| | | |
Line 195: |
Line 236: |
| <h2 style="color:purple">References</h2> | | <h2 style="color:purple">References</h2> |
| </div> | | </div> |
− | <div class="post-content"><p align="justify" <i> Extraction of Plant DNA by Microneedle Patchfor Rapid Detection of Plant Diseases, June 9, 2019, Rajesh Paul, Amanda C. Saville et al.</i>, <cite><a href="https://pubs.acs.org/doi/pdf/10.1021/acsnano.9b00193">[link to paper]</a></cite> </p> | + | <div class="post-content"><p align="justify" <i> Extraction of Plant DNA by Microneedle Patch for Rapid Detection of Plant Diseases, June 9, 2019, Rajesh Paul, Amanda C. Saville et al.</i>, <cite><a href="https://pubs.acs.org/doi/pdf/10.1021/acsnano.9b00193">[link to paper]</a></cite> </p> |
| | | |
| </p> | | </p> |