Fabrication of a Microneedle Patch
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.
FIGURE - A picture of one of our Microneedle patches.
MN Patch-Based DNA Extraction
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.
FIGURE - A picture of a leaf after application of a Microneedle patch. The extraction was performed three weeks before (left); one week before (right).
DNA Extraction with the DNeasy Plant Pro Kit
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.
After the extraction, all amplifications were performed by Polymerase Chain Reaction (PCR) using 1 µL of DNA , 25 µL of Q5 master mix and 2,5 µL of each primer in a 50 µL PCR reaction. We used EC_PCR_fwd/rev primer to amplify an amplicons of 214 bp of the vine gene.
After amplification, gel electrophoresis was performed to visualize the amplified PCR products. For that, agarose, 10× (TE) buffer, midori green gel stain ,gel pilot 6X loading dye,5 µL GelPilot 50 bp l DNA adder (Qiagen)were used. All PCR-amplified products were visualized in 2% agarose gel.