|
|
(6 intermediate revisions by 2 users not shown) |
Line 128: |
Line 128: |
| 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" > | | <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" > | | <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"> |
| The protocol consists of multiple steps. Samples are added to the tissue disruption tube, which contains a specially shaped bead and a buffer for rapid homogenization. Cell lysis and DNA release occur by mechanical and chemical methods. Released genomic DNA is cleared of PCR inhibitors using QIAGEN’s second-generation IRT and then captured on a silica membrane in a spin column format. DNA is then washed and eluted from the membrane and is ready for PCR and other downstream applications. </p> | | The protocol consists of multiple steps. Samples are added to the tissue disruption tube, which contains a specially shaped bead and a buffer for rapid homogenization. Cell lysis and DNA release occur by mechanical and chemical methods. Released genomic DNA is cleared of PCR inhibitors using QIAGEN’s second-generation IRT and then captured on a silica membrane in a spin column format. DNA is then washed and eluted from the membrane and is ready for PCR and other downstream applications. </p> |
Line 171: |
Line 175: |
| <article class="post"> | | <article class="post"> |
| <div class="post-wrapper"> | | <div class="post-wrapper"> |
− | <div class="post-header">
| |
| | | |
| <h5 style="color:indigo" id="spectrophotometer">Nanodrop analysis </h5> | | <h5 style="color:indigo" id="spectrophotometer">Nanodrop analysis </h5> |
− | </div>
| + | |
| | | |
| | | |
| | | |
| | | |
− | <img src="https://static.igem.org/mediawiki/2019/d/de/T--EPFL--PCR_MN_EC.jpg" >
| + | |
| | | |
| | | |
Line 186: |
Line 189: |
| | | |
| <div class="post-content"><p style="font-size:17px;" align="justify"> | | <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) | + | 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> |
− | 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> |
− | As expected, the negative control absorption at 260nm is close to zero, no DNA is present in the microneedle patch.
| + | 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. |
− | In contrast, the positive control has the typical DNA Nanodrop readout. </br>
| + | |
− | The DNA extracted by microneedle patch shows significant absorption at A260. Its line higher than the negative
| + | |
− | control but lower than the positive control. This means that some DNA was extracted but that it is not pure,
| + | <img src="https://static.igem.org/mediawiki/2019/2/22/T--EPFL--courbes.png" > |
− | which is normal since, as mentioned previously, the patch extracts DNA and other molecules on the leaf.
| + | </br> </p> |
− | </p>
| + | |
− |
| + | |
| <h5 style="color:indigo" id="Gel">PCR amplification</h5> | | <h5 style="color:indigo" id="Gel">PCR amplification</h5> |
− | <img src="https://static.igem.org/mediawiki/2019/c/c9/T--EPFL--3courbes.png" >
| |
| <p style="font-size:17px; align="justify"> | | <p style="font-size:17px; align="justify"> |
| We amplified different EC DNA by PCR and ran a gel electrophoresis to analyze the results. | | We amplified different EC DNA by PCR and ran a gel electrophoresis to analyze the results. |
− | We loaded the gel from left to right as following, the ladder, synthetic EC DNA sequence,
| |
− | DNA extracted with a microneedle patch, DNA extracted with a standard kit, control with primers but no DNA.
| |
| Bands are observed in all the lanes containing DNA, no band is present in the control. | | 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. | | This shows that the microneedle patch successfully extracted DNA from the plant, which was then amplified by PCR. |
| </p> | | </p> |
| | | |
− | | + | <img src="https://static.igem.org/mediawiki/2019/d/de/T--EPFL--PCR_MN_EC.jpg" > |
| + | |
| | | |
| | | |